Science topic

Molecular Biological Techniques - Science topic

Explore the latest questions and answers in Molecular Biological Techniques, and find Molecular Biological Techniques experts.
Questions related to Molecular Biological Techniques
  • asked a question related to Molecular Biological Techniques
Question
12 answers
My streptavidin ELISA is producing very high OD values for both my positive and negative sera. I have purchased commercially available streptavidin coated ELISA plates that have been pre-blocked. The protocol I am using is:
Initially washing the plates - incubating with biotinylated peptides (2 hrs) - blocking with PBS + 2% BSA (1 hr) - addition of primary Ab (bovine) diluted in PBS (1 hr) - addition of secondary Ab (rabbit anti bovine) diluted in PBS (1 hr) - each of these steps were performed at 37 degrees C and washing was performed between each step using 0.0125% Tween 20 in Saline.
Are there any suggestions on how can I reduce the high background observed across the plate and especially in my negative control sera?
Thanks
Relevant answer
Answer
Hi,
Blocking with PBS + 2% BSA (1 hr) must be before incubating with biotinylated peptides.
  • asked a question related to Molecular Biological Techniques
Question
1 answer
Does anyone have a tried and tested protocol for intracellular staining of RBC's. I have tried a few methods, but the RBC's all seem to disappear during the staining step!
Relevant answer
Answer
I also come up with this problem. If anyone find solution, please let others know!
  • asked a question related to Molecular Biological Techniques
Question
6 answers
I ligated a DNA size of 3.8kb into topo TA vector. I expected a band size of about 7.7kb from my agarose gel electrophoresis after plasmid purification but got a band size of about 5kb. Pls what does this band size signify or is topo vector less than 3kb?
Relevant answer
Answer
I hope you are doing well.
Yes, flow cytometry, commonly known as Fluorescence-Activated Cell Sorting (FACS), has been widely used on CRISPR-Cas9 transfected cells, and it serves as a powerful tool for the analysis and sorting of these modified cells. The application of FACS in this context generally focuses on the identification, quantification, and isolation of cells that have been successfully edited by CRISPR-Cas9.
Here's a detailed explanation of how FACS is utilized with CRISPR-Cas9 transfected cells:
  1. Detection of Transfection Efficiency: After transfecting cells with CRISPR-Cas9 components, such as Cas9 protein and guide RNA (gRNA), a fluorescent reporter (e.g., GFP or RFP) is often co-expressed. FACS is then used to detect the expression of these reporters, providing a reliable measure of transfection efficiency.
  2. Sorting of Edited Cells: For targeted gene editing, cells that exhibit specific phenotypic changes, such as the knockout or knock-in of a gene, can be selectively sorted using FACS. This approach allows for the enrichment of cell populations with the desired genetic modifications, improving the efficiency of subsequent experiments.
  3. Validation of Gene Editing: Beyond sorting, FACS can be employed to validate the functional outcomes of CRISPR-Cas9 editing. For example, if the target gene affects surface markers or other cellular characteristics detectable by flow cytometry, FACS can confirm successful editing at the functional level.
  4. Single-Cell Analysis: FACS allows for the isolation of single cells, which can be particularly valuable when generating clonal populations from edited cells. Single-cell analysis enables researchers to assess the heterogeneity of gene editing outcomes, which is critical for applications requiring precise genetic modifications.
These applications underscore the versatility of FACS in conjunction with CRISPR-Cas9 technology, providing both qualitative and quantitative insights into the gene editing process. Proper optimization of FACS parameters, including antibody selection, gating strategies, and fluorescence compensation, is essential for achieving accurate results.
If you have any specific questions or require further details, please do not hesitate to reach out.
This list of protocols might help us better address the issue.
  • asked a question related to Molecular Biological Techniques
Question
3 answers
I'm using the CRISPR/Cas9 gene editing method to target a gene in human iPSCs for mutation correction via the homology directed repair pathway. I want to compare the efficiency of different gRNAs to determine which gRNA will be the best to use for this application. Normally, this can easily be done using a genomic cleavage detection kit or a mismatch detecting endonuclease, followed by visualization of the resulting fragments on a gel; however, the mutation we are trying to correct is heterozygous in this particular patient. Since mismatch endonucleases, such as the surveyor nuclease, are sensitive enough to detect a single bp change, would the heterozygous nature of this SNP lead to a overestimate of the level of cleavage occurring? 
Has anyone else come across this issue? If so, does anyone have suggestions for other methods to detect cleavage events?
Relevant answer
Answer
Dear Colleague,
I hope this message finds you well. Detecting CRISPR/Cas9-induced genomic cleavage in heterozygous cell lines involves several steps to identify and confirm the presence of mutations. Here is a detailed and logical approach to detect these genomic changes:
Steps for Detecting CRISPR/Cas9-Induced Genomic Cleavage
  1. Design and Delivery of CRISPR/Cas9 Components:sgRNA Design: Ensure that the single guide RNA (sgRNA) is designed to target the specific genomic region of interest. CRISPR/Cas9 Delivery: Use a suitable delivery method (e.g., plasmid transfection, ribonucleoprotein complexes) to introduce the Cas9 and sgRNA into the heterozygous cell line.
  2. Cell Culture and DNA Extraction:Cell Culture: Grow the transfected cells under appropriate conditions. Allow sufficient time for the CRISPR/Cas9 system to induce cleavage and for cells to repair the induced double-strand breaks. DNA Extraction: Extract genomic DNA from the edited cell population using a standard DNA extraction kit or protocol.
  3. PCR Amplification of Target Region:Primer Design: Design primers flanking the CRISPR target site to amplify the region of interest. PCR Conditions: Optimize PCR conditions to ensure specific and efficient amplification. Use high-fidelity DNA polymerase to minimize errors during amplification.
  4. Detection of Indels (Insertions/Deletions): T7 Endonuclease I (T7E1) Assay: This enzyme detects mismatches in heteroduplex DNA formed by hybridizing wild-type and edited DNA strands.Denature and reanneal the PCR product to form heteroduplexes. Treat with T7E1 and analyze the digestion products on an agarose gel. Surveyor Nuclease Assay: Similar to T7E1, this enzyme cleaves mismatched DNA and can be used to detect indels. High-Resolution Melt (HRM) Analysis: Detects differences in melting curves of PCR products from wild-type and edited DNA. Restriction Fragment Length Polymorphism (RFLP): If the edit creates or destroys a restriction enzyme site, digest the PCR product and analyze the pattern on a gel.
  5. Sequencing for Confirmation: Sanger Sequencing: Sequence the PCR products to identify and confirm the exact nature of the edits.Clone the PCR products into a TA cloning vector and sequence individual clones to separate different alleles. Next-Generation Sequencing (NGS): Use NGS for high-throughput and detailed analysis of the edited region.Prepare an amplicon library and sequence to detect and quantify all possible mutations.
  6. Analysis and Interpretation:Quantification of Editing Efficiency: Analyze the sequencing data to determine the frequency and types of mutations introduced. Bioinformatics Tools: Use tools like CRISPResso or TIDE (Tracking of Indels by Decomposition) to analyze sequencing data and quantify editing outcomes.
Example Protocol:
  1. Design and Transfection:Design sgRNA and transfect cells with CRISPR/Cas9 plasmid or RNP complex.
  2. DNA Extraction:Extract genomic DNA 48-72 hours post-transfection.
  3. PCR Amplification:Design primers flanking the target site and perform PCR amplification.
  4. Mutation Detection: T7E1 Assay:Denature and reanneal PCR products. Treat with T7E1 and run on an agarose gel. Sanger Sequencing:Clone PCR products and sequence individual clones. NGS:Prepare and sequence an amplicon library.
  5. Data Analysis:Use bioinformatics tools to analyze and quantify mutations.
By following these steps, you can effectively detect and confirm CRISPR/Cas9-induced genomic cleavage in heterozygous cell lines.
Should you have any further questions or require additional assistance, please feel free to reach out.
Perhaps this protocol list can give us more information to help solve the problem.
  • asked a question related to Molecular Biological Techniques
Question
5 answers
I infected the cell lines H1437, H2073 and H2228 with a lentivirus that express resistance to puromycin. Does anyone knows the best dosis and time for selection? I have found information using 2 micrograms per ml for 3 days, but when I did the kill curve for H2228 it did not seem to be enough.
Any information or experiences would be greatly appreciated!
Thank you!
Relevant answer
Answer
Dear Colleague,
I hope this message finds you well. Determining the optimal puromycin concentration for selecting non-small cell lung cancer (NSCLC) cell lines transformed with lentivirus requires an initial kill curve experiment to identify the lowest concentration that effectively kills untransfected cells while sparing the transfected ones. Here is a detailed and logical approach to determining the best puromycin concentration:
Steps to Determine the Optimal Puromycin Concentration
  1. Prepare Cells:Cell Seeding: Plate NSCLC cells in a 24-well or 6-well plate at a density that allows them to reach 70-80% confluency within 24 hours. This ensures they are actively dividing and will respond uniformly to puromycin.
  2. Prepare Puromycin Stock Solution:Stock Solution: Prepare a stock solution of puromycin (e.g., 10 mg/mL) by dissolving puromycin in sterile water. Filter-sterilize the solution and store aliquots at -20°C.
  3. Perform Kill Curve:Dilution Series: Prepare a dilution series of puromycin in the culture medium (e.g., 0, 0.5, 1, 2, 4, 6, 8, 10 µg/mL). Add the different concentrations to the wells containing NSCLC cells. Incubation: Incubate the cells with puromycin for 3-7 days, refreshing the medium with puromycin every 2-3 days. Monitoring: Observe cell viability daily using a microscope. Identify the lowest concentration of puromycin that results in complete cell death (no viable cells remaining) within this period.
  4. Selection of Transduced Cells:Transduction: Infect NSCLC cells with the lentivirus carrying the puromycin resistance gene. Ensure a high multiplicity of infection (MOI) to increase the efficiency of transduction. Post-Transduction Recovery: Allow the cells to recover for 24-48 hours post-transduction before applying puromycin. Puromycin Selection: Apply the previously determined optimal puromycin concentration (typically between 1-10 µg/mL) to the culture medium to select for transduced cells. Monitoring: Replace the medium with fresh puromycin-containing medium every 2-3 days and continue selection until non-transduced cells are completely eliminated (usually 3-7 days).
  5. Validation:Efficiency Check: Validate the selection efficiency by assessing the expression of the transgene or marker gene carried by the lentivirus. This can be done using methods such as qPCR, Western blotting, or fluorescence microscopy if a fluorescent marker is present.
Example Protocol
  1. Cell Seeding: Seed NSCLC cells at 50,000 cells per well in a 24-well plate.
  2. Puromycin Dilution Series: Prepare puromycin concentrations of 0, 0.5, 1, 2, 4, 6, 8, and 10 µg/mL.
  3. Application and Monitoring: Apply the puromycin series and monitor cell death daily.
  4. Optimal Concentration: Determine the lowest concentration that kills all non-transduced cells within 3-7 days.
  5. Transduction and Selection:Infect cells with lentivirus. Allow recovery for 24-48 hours. Apply the optimal puromycin concentration for selection.
By following these steps, you can accurately determine the best puromycin concentration for selecting NSCLC cell lines transformed with lentivirus, ensuring effective selection of transduced cells while minimizing toxicity.
Should you have any further questions or require additional assistance, please feel free to reach out.
This protocol list might provide further insights to address this issue.
  • asked a question related to Molecular Biological Techniques
Question
6 answers
We are trying to clone into a plasmid(pBUDce4.1) with Zeocin resistance, but are finding it very difficult. The Zeocin appears to have very mild selectivity (i.e. transformed cells grow only slightly faster than untransformed cells on Zeocin agar plates). We have now managed to select transformed colonies, but when we grow up liquid cultures there is very little plasmid post extraction on agaros gel .Is this poor selectivity something seen by other people (I am aware Zeocin is unstable in high salt, light etc. so I am using low salt (5mg/l) LB and trying to keep out of direct light)? Any other thoughts as to why this isn't working for us?
Relevant answer
Dear S. Sharif
I am struggling with a similar problem, my untransformed cells are growing on Zeocin plates. How you manage to solve the problem?
Thank you
  • asked a question related to Molecular Biological Techniques
Question
4 answers
My sample is filtered and concentrated liquid and I'm using Qiaamp viral RNA mini kit to elute viral RNA.
Since this kit is not designed to separate viral RNA from cellular DNA I used RNase-free DNase set (Qiagen) for DNase treatment. I pour 80ul of DNase to the column for 15min. at room temperature before the washstep (AW1 and AW2).
260/280 of the eluted RNA was too high.. it was almost 3.4.. and it  is also very high even when I didn't do DNase treatment.. I have no idea why this kind of result shows up.
Relevant answer
Answer
안녕하세요. 옛날 글에 이렇게 댓글 남기게 되어 죄송합니다. 혹시 해결법을 찾으셨나요?
  • asked a question related to Molecular Biological Techniques
Question
3 answers
I will be running my first luciferase assay and the protocol I am using does not specify what filters or filter settings I should use. The plate will be read on a lumometer from Biotek, Synergy HT. What would a general filter setting be to measure the luminescence?
Relevant answer
Answer
Hello Dear Cody,
This is my question too. I am also working with Luciferase for the first time and I have questions about the plate, the device and the wavelengths, but the answers to your questions cannot be found here. Please tell me the answer too.
  • asked a question related to Molecular Biological Techniques
Question
2 answers
Does anyone know how to make the fluid to remove the coverslip of glass bottom dishes?
Relevant answer
Answer
Dear Ayni Sharif,
I had similar problem recently and after some experiments I found that you can use either isopropanol (15h incubation) or WD-40 (5-6h incubation). I poured this agents at the bottom of bigger dish and placed glass bottom dish inside. So liquids come in touch with outside surface of glass bottom dish. After incubation (at 25 degree) you can wipe glass bottom from outside and gently push the glass (with samples) out.
Hope it helps.
  • asked a question related to Molecular Biological Techniques
Question
5 answers
I am performing a KO of my gene by frameshift in zebrafish. I have been screening this by the disruption of a restriction enzyme site in a PCR fragment. I now have an incross of 2 mutant fish and am hoping raise the progeny and get some homozygous fish. I will use DNA from tissue sample as the PCR template but I am wondering how I can separate homozygous from heterozygous. In my experience so far the F1 generation from an outcross with wild type showed an extremely faint undigested band (wt DNA) compared to the really bright digested band (indicating mut DNA) for some reason even though an outcross should automatically result in heterozygous fish containing both wt & mut (digested & undigested) DNA. I presumed that the concentration of the mut & wt allele would be similar. Has anyone come across this difficulty before. Could there be a reason a mutant would amplify more in a PCR reaction (it is only 7 bp shorter)? Any suggestions for a way to tell the difference? I am concerned since the band is so faint on heterozygotes I may misidentify heterozygotes as homozygotes.
Relevant answer
Answer
Dear Esteemed Colleague,
Greetings. I trust this message finds you well and engrossed in the cutting-edge realm of genome editing, particularly employing CRISPR-Cas9 technology. Your inquiry about separating homozygous from heterozygous mutations post-CRISPR is both crucial and timely for ensuring the precision and efficacy of genetic modifications. Below, I provide a comprehensive guide outlining methodologies for distinguishing between these genetic variants, thereby enhancing the resolution of your CRISPR-based experiments.
Overview
The CRISPR-Cas9 system enables targeted genomic alterations with high specificity. Following the introduction of CRISPR-Cas9 components into cells, the repair of the induced double-strand break can result in modifications leading to homozygous or heterozygous alleles. Determining the zygosity of these modifications is essential for characterizing the functional consequences of the edits.
Methodologies for Zygosity Determination
  1. PCR and Sanger Sequencing:Procedure: Amplify the target region surrounding the CRISPR-induced modification using PCR, followed by Sanger sequencing of the PCR products. Analysis: Compare the sequencing chromatogram of the edited sample to that of a wild-type control. Homozygous mutations will show a single, clean peak at the modification site, whereas heterozygous mutations will display overlapping peaks, indicating the presence of both the wild-type and edited alleles.
  2. Restriction Fragment Length Polymorphism (RFLP) Analysis:Procedure: If the CRISPR edit introduces or abolishes a restriction enzyme site, perform a PCR to amplify the target region, followed by digestion with the appropriate restriction enzyme. Analysis: Resolve the digested PCR products on an agarose gel. The pattern of bands will differ between homozygous and heterozygous alleles based on the presence or absence of the restriction site.
  3. T7 Endonuclease I (T7EI) Assay:Procedure: Amplify the target region via PCR and denature and reanneal the PCR products to form mismatched duplexes if both edited and wild-type alleles are present. Treat the reannealed products with T7 Endonuclease I, which cleaves mismatched DNA. Analysis: Homozygous edits will not generate cleavage products, whereas heterozygous edits will result in a cleavage pattern visible on an agarose gel, indicating the presence of mismatches.
  4. Digital Droplet PCR (ddPCR):Procedure: Design allele-specific probes for ddPCR that can distinguish between the wild-type and edited alleles. Analysis: Quantify the absolute number of edited and wild-type alleles. The ratio of these alleles can precisely determine zygosity, with a 1:1 ratio indicating heterozygosity and a predominance of one allele type indicating homozygosity.
  5. Next-Generation Sequencing (NGS):Procedure: Perform deep sequencing of the target region in a population of cells. Analysis: Bioinformatic analysis can quantify the proportion of reads representing the wild-type versus edited alleles, providing a detailed assessment of zygosity at the population level. For clonal populations, NGS can definitively characterize the zygosity of individual clones.
Considerations and Best Practices
  • Clonal Isolation: For precise zygosity determination, especially in heterogeneous populations, isolating single clones and expanding them for individual analysis is recommended.
  • Validation: Confirm the editing and zygosity results using multiple, complementary methods when possible to ensure accuracy.
  • Bioinformatic Support: Leverage bioinformatics tools for analyzing complex sequencing data, particularly when using high-throughput methods like NGS.
By employing these strategies, you can accurately determine the zygosity of CRISPR-induced genetic modifications, a critical step in validating the success of your genome editing endeavors and understanding the functional implications of your edits.
Should you require further assistance or wish to delve deeper into any of these methodologies, please do not hesitate to reach out. I am here to support your research journey and contribute to the advancement of genome editing technologies.
Warm regards.
Take a look at this protocol list; it could assist in understanding and solving the problem.
  • asked a question related to Molecular Biological Techniques
Question
4 answers
Hi All, I am transfecting two vectors (one gRNA vector and a HDR vector) to KO a gene. I have puromycin resistant in HDR vector. I am using Fugene for transfection purpose. I was wondering to know how long should I wait to start selection after transfection of the crispr vectors?
Relevant answer
Answer
The timing for starting selection after transfection of CRISPR vectors can depend on various factors, including the specific experimental setup, the type of CRISPR system used, and the characteristics of the target cells. However, here are some general considerations to help you determine when to initiate selection after transfection:
  1. Transfection Efficiency: Allow sufficient time for the CRISPR vectors to be transfected into the target cells and express the components necessary for genome editing (e.g., Cas9 enzyme, sgRNA). Transfection efficiency can vary depending on the transfection method and cell type, but it typically ranges from a few hours to a day.
  2. Expression of CRISPR Components: The timing for initiating selection should ensure that the CRISPR components have had enough time to be expressed and become functional within the cells. This can range from several hours to a day or more, depending on the kinetics of gene expression and protein production in the specific cell type.
  3. Off-Target Effects: Initiating selection too soon after transfection may increase the risk of off-target effects, where the CRISPR system induces unintended mutations at genomic loci with partial sequence homology to the target site. Allowing sufficient time for specific on-target editing to occur before applying selection pressure can help minimize off-target effects.
  4. Cell Proliferation Rate: Consider the proliferation rate of the target cells when determining the timing of selection. Cells with faster proliferation rates may require shorter selection times compared to cells with slower proliferation rates to achieve the desired level of genome editing.
  5. Optimization: Perform pilot experiments or optimization studies to determine the optimal timing for selection in your specific experimental system. Test different time points for initiating selection and assess the efficiency and specificity of genome editing under each condition.
  6. Balancing Selection Pressure and Cell Viability: Initiate selection early enough to exert adequate selection pressure on cells containing the CRISPR-mediated edits while ensuring that cell viability is not compromised. Too long a delay in starting selection may allow unedited or less efficiently edited cells to outgrow edited cells.
Based on these considerations, it's common to start selection for edited cells within 24-48 hours after transfection of CRISPR vectors. However, the optimal timing may vary depending on the specific experimental setup and cell type. It's essential to empirically determine the best timing for selection in your particular experimental context through pilot experiments and optimization studies.
l This list of protocols might help us better address the issue.
  • asked a question related to Molecular Biological Techniques
Question
9 answers
I am planning to work with Addgene's pLKO-tet-on plasmid. I checked the protocol on the website and comments left here so far. I am a bit confused. Should I design the oligos exactly in the protocol (5'-CCGG for AgeI and 5'-AATT for EcoRI)? Because it is one bp missing in both RE sites and it results in oligos which have mutant RE sites. If they are mutant, how will the ligation work? I would be really happy if someone could explain me. One more thing, I came across so many people who had troubles of getting positive clones after ligation. I am wondering what is the latest situation. Is there any tricks that I should know? Thank you very much for your answers in advance.
Relevant answer
Answer
Designing shRNA (short hairpin RNA) oligonucleotides for the pLKO-tet-on plasmid involves several steps to ensure specificity, efficiency, and compatibility with the vector system. The pLKO-tet-on system allows for doxycycline-inducible expression of shRNA, offering controlled gene silencing. Here's a general guide on how to design shRNA oligos for this system:
1. Target Gene Selection
  • Choose the gene you wish to silence. It's crucial to select a target sequence that is unique to your gene of interest to avoid off-target effects.
2. shRNA Sequence Design
  • Target Sequence Length: Typically, 19-21 nucleotides long sequences within the target mRNA are chosen for shRNA design.
  • GC Content: Aim for a GC content of 40-60% to ensure stable shRNA formation and efficient target binding.
  • Blast Search: Use the NCBI BLAST tool to check the specificity of your chosen sequence against the genome of your organism to avoid off-target silencing.
  • Avoid Polypurine Stretches: Sequences with long stretches of Gs or Cs can form strong secondary structures or promote Pol III termination, reducing shRNA efficiency.
3. Loop Sequence
  • A loop sequence is required to connect the sense and antisense strands of the shRNA. A commonly used loop sequence is TTCAAGAGA, but others can also be effective.
4. Termination Signal
  • A string of 5-6 thymidines (T's) acts as a Pol III termination signal and should be added at the end of the antisense strand.
5. Incorporating Overhangs for Cloning
  • The pLKO-tet-on vector uses specific restriction sites for cloning (commonly AgeI and EcoRI). You'll need to add 5' overhangs compatible with these sites to your oligos. For example:Forward oligo: 5'-CCGG(N19 sense sequence)TTCAAGAGA(N19 antisense sequence)TTTTTG-3' Reverse oligo: AATTCAAAAA(N19 reverse complement of antisense sequence)TCTCTTGAA(N19 reverse complement of sense sequence)-3'
  • The CCGG and AATT sequences at the ends of the oligos correspond to the overhangs created by AgeI and EcoRI digestion, respectively.
6. Oligo Synthesis and Cloning
  • Have your designed oligos synthesized by a reputable company. Upon receiving, anneal the oligos to form double-stranded DNA and ligate into the digested pLKO-tet-on vector.
7. Validation
  • After cloning, sequence verify the insert to ensure correct shRNA sequence integration. It's also important to test the efficiency of gene knockdown by your shRNA in a pilot experiment before proceeding with more extensive studies.
8. Inducibility Test
  • Verify the inducibility of your shRNA expression by treating transfected cells with and without doxycycline and measuring the target gene's mRNA and protein levels.
Designing effective shRNA oligos requires careful consideration of the target sequence, shRNA structure, and cloning strategy. The pLKO-tet-on system's inducible nature adds an extra layer of control to your gene silencing experiments, making it a powerful tool for studying gene function.
l Perhaps this protocol list can give us more information to help solve the problem.
  • asked a question related to Molecular Biological Techniques
Question
5 answers
I have generated the lentivirus particles with my GOI. But  did not get transduction efficiency in Jurkat cells post 72 hrs why? any suggestion ?
Particulas: used 24 well plate :
50,000 cells with Polybrene 8ug/ml
Thank you in anticipation
Relevant answer
Answer
Lentiviral transduction is a widely used method for introducing foreign genes into various cell types, including hard-to-transfect cell lines like Jurkat cells. Jurkat cells, a human T lymphocyte cell line, are commonly used in research on T-cell signaling and function. Here are some tips and considerations for successful lentiviral transduction in Jurkat cells based on common practices and literature:
Optimize Multiplicity of Infection (MOI)
  • Multiplicity of Infection (MOI) refers to the ratio of viral particles to target cells. For Jurkat cells, starting with an MOI of 5-10 is common, but this can vary based on the viral titer and the gene of interest. Optimization experiments to find the best MOI for your specific construct and goals are essential.
Use Polybrene
  • Polybrene (hexadimethrine bromide) is a cationic polymer used to enhance viral transduction efficiency by neutralizing charge interactions between the cell surface and viral particles. A final concentration of 4-8 µg/mL is typically used, but high concentrations can be toxic to cells, so optimization is necessary.
Spinoculation
  • Spinoculation (centrifugal transduction) can improve transduction efficiency. This involves centrifuging the cells with the viral supernatant at low speeds (e.g., 800-1,000 g for 1-2 hours at 32°C). However, the optimal conditions (speed, temperature, and duration) should be determined experimentally.
Incubation Time
  • After adding the virus, cells are usually incubated for several hours to overnight to allow for sufficient viral entry. Post-transduction, cells can be refreshed with new media to remove viral particles and polybrene, reducing toxicity.
Selecting Transduced Cells
  • If your lentiviral vector includes a selectable marker (e.g., puromycin resistance), applying the selection drug 48-72 hours post-transduction can help enrich for transduced cells. The optimal concentration and duration of selection should be determined for Jurkat cells in advance.
Assessing Transduction Efficiency
  • Transduction efficiency can be evaluated by flow cytometry if the vector expresses a fluorescent marker (e.g., GFP) or through PCR, Western blot, or functional assays for your gene of interest.
Considerations
  • Jurkat cells are suspension cells, making certain steps (like changing media post-transduction) slightly different from adherent cell protocols.
  • Ensure that the lentiviral vector's promoter is active in T-cells. Some promoters (like CMV) can be silenced in certain cell types.
  • Be aware of biosafety considerations when working with lentiviral vectors. Generally, lentiviral work is conducted under BSL-2 conditions.
Tips for Success
  • Always start with a pilot experiment to optimize the MOI and polybrene concentration.
  • Consider using a lentiviral vector with a reporter gene to easily monitor transduction efficiency and optimize conditions before using your experimental vector.
  • Ensure your lentiviral preparation is of high quality and titer. Poor-quality virus can lead to low transduction efficiency and high cell death.
Collaborating with a lab experienced in lentiviral transduction or consulting the literature for protocols specific to Jurkat cells can also provide valuable insights and help troubleshoot common issues.
l This list of protocols might help us better address the issue.
  • asked a question related to Molecular Biological Techniques
Question
6 answers
I am working on unknown large plasmids (50-500 kb) that I need to characterize. I plan to use the Large Construct kit for extracting them and remove chromosomal DNA.
1) Will I see large plasmids on a gel???
2) Is Primer Walking a good approach?
Is there any other methods less time consuming?
NGS will be done but I want to confirm using Sanger Sequencing.
Thanks,
Relevant answer
Answer
Sequencing large plasmids, which can range from a few thousand to over a hundred thousand base pairs, presents a challenge due to their size and complexity. However, there are several effective methods for sequencing large plasmids:
  1. Next-Generation Sequencing (NGS):Illumina Sequencing: This technique provides high-throughput, accurate, and cost-effective sequencing. It involves fragmenting the plasmid DNA, sequencing these fragments, and then assembling the sequences using bioinformatics tools. However, repetitive regions can be challenging to assemble. Long-Read Sequencing (PacBio and Oxford Nanopore): These technologies generate longer reads, which are particularly useful for sequencing large plasmids and resolving complex regions. PacBio's SMRT sequencing and Oxford Nanopore's technology can produce reads over 10,000 base pairs long, aiding in spanning repetitive sequences and simplifying assembly.
  2. Sanger Sequencing:While this method is more traditional and has lower throughput, it is highly accurate for shorter DNA sequences. For large plasmids, a strategy of 'primer walking' can be used, where sequential rounds of sequencing are conducted, each starting where the last one left off.
  3. Hybrid Approaches:Combining short-read (e.g., Illumina) and long-read (e.g., PacBio or Nanopore) sequencing can provide both the accuracy of short-read sequencing and the long-read ability to span complex regions. This approach can effectively resolve the structure of large plasmids.
  4. Optical Mapping:Technologies like BioNano Genomics provide large-scale, high-resolution mapping of DNA. While not sequencing per se, optical mapping can help in assembling and validating the plasmid structure, especially useful for very large or complex plasmids.
The choice of method depends on the specific requirements of the project, such as the size of the plasmid, the complexity of its sequence (like the presence of repetitive elements), the required accuracy, and the available budget. For most purposes, a combination of high-throughput short-read sequencing for accuracy and long-read sequencing for spanning complex regions provides a comprehensive approach to sequencing large plasmids.
l Check out this protocol list; it might provide additional insights for resolving the issue.
  • asked a question related to Molecular Biological Techniques
Question
4 answers
I've been trying to perform a Northern blot for a while now, with no positive results.
I use Digoxigenin labeled PCR products as a probe (after boiling for 5min) in hybridization buffer (50%formamide + 5xSSC, 50mM Na-P, 1%SDS, 10ugssDNA/ml, 5xDenharts) @42*C o/n, dollowed by wash, block in 2%milk 0.3% PBS-T, and incubation with an anti-Digoxin antibody.
I know the transfer works, and that RNA is present after prehybridization (via methylene blue), I also know the antibody works due to dot blotting the DIG-labeled probes.
Does methylene blue staining inhibit probe binding?
Every time Ive tried N.blot Ive stained my membrane with methyl blue before in order to see whether the transfer worked. It's all I can think of at this point, otherwise it has to be my probes.
-CP
Relevant answer
Answer
Yes, the MeBlue had no real effect. The issue was due to my blocking reagent. I had been using 5% milk (because I'd adapted my Southern Blot protocol). After switching to Maleic Acid Blocking buffer, everything worked.
Maleic Acid Blocking buffer (0.1M Maleic Acid; 0.15M NaCl2) + 10%w/v Roche Blocking reagent (10X)- Maleic Acid made first, filtered. 10% w/v Blocking reagent added and mixed via microwave, then autoclaved. This is 10X so dilute 1:10 for blocking steps.
I've attached my protocol in case anyone needs it.
  • asked a question related to Molecular Biological Techniques
Question
4 answers
I am analyzing samples transfected by CRISPR using the T7 assay. And when I am trying to analyze it on the gel, I can found smears on my samples. I don't know if I added too much enzyme or very long incubation time. Any suggestions?
Here is my recipe/protocol used.
purified PCR product -150ng
NEB buffer 2- 1uL
Water- 9.5uL
Reaction 95C-10min, 85C- 5min (0.1C/sec), 65C-2min (0.1C/sec), 45C-2min (0.1C/sec), 25C-hold
Add 0.5uL T7 enzyme. Incubate for 1hr. Stop reaction by adding 1.5uL 0.25M EDTA. Run on gel.
thank you.
Reaction: 
Relevant answer
Answer
Hello,
Achieving clean and distinct bands in a gel after a T7 Endonuclease I assay, which is commonly used for detecting CRISPR-Cas9 induced mutations, requires careful attention to various steps in the procedure. Smearing can often be a challenge, but here are some effective strategies to minimize it:
  1. Optimal Digestion Conditions: Ensure that the T7 Endonuclease I digestion is performed under optimal conditions. This includes the correct buffer, enzyme concentration, and incubation time and temperature. Over-digestion can lead to smearing, so it's crucial to find the right balance.
  2. Quality of DNA: Use high-quality, purified DNA for the assay. Impurities in DNA samples can inhibit enzyme activity or interfere with electrophoresis, leading to smears.
  3. Gel Concentration: Choose the appropriate agarose gel concentration. A higher percentage gel can resolve smaller fragments more effectively, which might be beneficial depending on the expected size of the digested products.
  4. Loading Buffer: Use an appropriate loading buffer and ensure that it is mixed thoroughly with your samples. This helps the DNA to enter and migrate through the gel evenly.
  5. Electrophoresis Conditions: Run the gel at a consistent and appropriate voltage. Higher voltages can cause the gel to heat up and lead to smearing. A slower run at a lower voltage often yields better resolution.
  6. Sample Volume: Avoid loading too much sample into the wells, as overloading can cause smearing. If necessary, load multiple wells with smaller volumes.
  7. Gel Loading Technique: Be careful while loading the samples into the wells. Pipetting gently and accurately is key to preventing smearing.
  8. Avoiding Contamination: Ensure that all pipettes, tubes, and tips are clean to avoid cross-contamination between samples.
  9. DNA Fragment Size: Be mindful of the expected size of the cleaved DNA fragments. If the fragments are very small, they might appear as a smear. Adjusting the gel concentration or running time can help resolve this issue.
  10. Post-Electrophoresis Handling: Handle the gel carefully after electrophoresis. Excessive handling or harsh staining/destaining procedures can cause smearing.
  11. Positive Controls: Include a positive control known to yield clear bands in the assay. This will help you determine if the smearing is a sample-specific issue or a more general problem with the assay conditions.
  12. Troubleshooting: If smearing persists, consider troubleshooting by varying one condition at a time, such as enzyme concentration, digestion time, or gel percentage.
By following these guidelines, you can minimize smearing in your gels after a T7 Endonuclease I assay, leading to clearer and more interpretable results. Remember, meticulous technique and optimization are often key to successful gel electrophoresis.
This protocol list might provide further insights to address this issue.
  • asked a question related to Molecular Biological Techniques
Question
2 answers
Is there any affinity of microRNA to glass surfaces? I tried to homogenize brain samples in order to get maximum yield of microRNA.
Relevant answer
Answer
the glass surface is likely to adsorb DNA/RNA, it's better to use some other beads such as ceramic or metal https://lab.plygenind.com/mastering-bead-selection-for-effective-homogenization
  • asked a question related to Molecular Biological Techniques
Question
6 answers
Hi I am looking for any alternative protocols to purify TMV virions, other than the commonly used PEG precipitation. 
Two main problems that I have with PEG protocol is that,
1\ any macromolecule will co-precipitate as well
2\ so that PEG cannot purify full length (300nm) TMV virions from other shorter rods (probably breakage), and other small discs. (thanks to people responded to my earlier question)
So I am wondering if there is any purification protocol or method that can get mostly the full length rods and rid breakage ones and discs? 
Thanks a lot in advance
Relevant answer
Answer
Ammonium Sulphate precipitation works and may help resolve the particles by size. Note that the salt itself may affect the stability of the particles. In my hands, a 45% cut contained TMV CP monomer and multimers up to ~300k( analysed with boiled, reduced SDS-PAGE). I have read elsewhere that a 15% saturation is enough to precipitate rods.
  • asked a question related to Molecular Biological Techniques
Question
4 answers
I am trying to isolate a nuclear protein NF-kB from raw cells in order to do a western blot.
I have searched some protocol for nuclear isolation and finally came up with one. But in that protocol, apart from adding 420mM Nacl for NE buffer , they have added an extra 400mM using 5M NaCl directly onto nuclear pellet and again 1 pellet volume of NE buffer. Can anyone tell why extra NaCl should be added, and if we add double the volume of NE buffer, won't the protein get diluted? I'm attaching that protocol along with this.
Relevant answer
Answer
Hi Sabina. According to the 1983 paper that described the preparation of NE from Hela cell, transcription looks good in 0.42 M NaCl. So the high salt buffer condition, 420 mM is adopted in most cases. Here is the paper Good luck!
  • asked a question related to Molecular Biological Techniques
Question
2 answers
i am new to enzymology and want to study the discipline through history based books (i,e, how was the Kreb's cycle discovered, methods used then, etc). what are your suggestions to achieve this?
better to be from wiley or springer.
Relevant answer
Answer
Arthur Kornberg, For the Love of Enzymes: The Odyssey of a Biochemist, is mainly focused on the discovery of enzymes involved in nucleic acid acid replication.
  • asked a question related to Molecular Biological Techniques
Question
6 answers
Hello, I have developed a Surface Plasmon resonance sensor using LED of wavelength 635nm and CMOS webcam as source. I am using the diverging rays of the LED as the change in incident angle. When I put silver coated glass slide on the prism I get a dip at a particular angle. I have test the sensor by immobilizing with MUA , EDC/NHS and IgG. The sensor can detect the shift in angle for all the layers. But when I put liquid dielectric medium like DI water, BSA or PBS buffer the shift disappears. I can monitor real-time data with the webcam and so when the liquid sample is passed I should be able to detect the shift. I have attached the file of how the dip looks like.
Relevant answer
Answer
Did you find the solution? I am in the same trouble.
  • asked a question related to Molecular Biological Techniques
Question
20 answers
I am having a rather odd issue with a ligation procedure. After a (hopefully) successful ligation of a 1kb insert into a 6kb vector, I transformed some Top 10 cells and got (very few) colonies on LB+Amp plates, but always more than the no-ligation control plates (indicating a hopefully successful ligation). I screened several (5-10) of these colonies and isolated DNA using a mini prep, after which I linearized all the DNA and ran on a gel. At first, I got really fuzzy bands, both in the samples and in the ladder, so it was hard to distinguish if they had any insert in them. Also, the unligated vector ran at a different size than expected, and some of the samples had two bands in them or ran faster than the unlighted vector. Overall, it seemed like a very messy gel, so I re-optimized my gel conditions to make sure I could at make any conclusions from the DNA sizes (made new buffer, made sure the loading was done correctly, lowered the voltage to use 90V for 2 hours using a 0.7% gel). After this, my ladder looked crisp and properly separated, and my unligated vector (linearized) ran at the right length and also looked pretty clear. However, all my ligated samples had absolutely no product in them at all! For the first gel, I used 500 ng per well, and all the wells looked equally bright and with similar amounts of DNA. Noticing this was a lot of DNA to run, I reduced it to 125ng of DNA, which showed up perfectly fine for the vector but not for the samples. There is no smear so I don't think it's degradation or nuclease contamination, so I am not sure what to do next. Any ideas? Thanks! I am attaching an image of the gel, with the only visible band being the linearized unligated vector (6kb), and all other wells being my ligation products. Any help would be greatly appreciated!
Relevant answer
Answer
I also had this problem. Before you run the gel, purify the ligation product to remove the buffer and the T4 ligase, then you will see the band.
  • asked a question related to Molecular Biological Techniques
Question
11 answers
Hi,
For the FreeStyle HEK 293F cell serum-free suspension culture, is it possible to use another media (a cheaper one) than the FreeStyle 293 Expression Medium? 
Thanks.
Relevant answer
Answer
So helpful the information is! Thank you!
  • asked a question related to Molecular Biological Techniques
Question
6 answers
I expect a 70 bp amplicon which shows up only with 2% (or higher) DMSO in the PCR. But with DMSO, the size of this amplicon goes from 70 bp to ~110b bp on 3% agarose gel. Same happens when a positive control PCR is done using same primers. In the positive control too, the size goes from 70 bp to ~110 bp when done with 2%DMSO. Is this size shift common if DMSO is added in to PCR?
Relevant answer
Answer
Hi,
I'm bringing up this post!
Sanjay Premi your sequencing results gave me some hope ... Let me explain, I try to amplify yan ITS target on grapevine DNA, expected amplicon size : variable around 200 and some.
Here is what I get
Without DMSO: non reproducible results between samples or between pcr on the same sample. In general: very weak amplification (bands not visible or very weak), one or more bands, very variable band sizes, some are > 500 bp.
With DMSO: VERY reproducible results between samples and PCR, ALWAYS only one very visible band slightly < 500 bp.
I have not done any sequencing for the moment.
Sanjay Premi if you still have pictures of your gels with and without DMSO, are you ok to share them with me? or do you simply know more about the origin of this?
Kind regards
Paola
  • asked a question related to Molecular Biological Techniques
Question
4 answers
Hi
I would like to ask about protein precipitation by salting out method.
I'm going to precipitate collagen from fish scales. Some articles said the supernatant of the extract salted out by adding NaCl to a certain concentration, for example 2.0 M. Does it mean adding solid NaCl or NaCl solution? And how to calculate it?
Can you explain it more detail?
I've read ebook from Coligan (2001), Current Protocols in Protein Science. The book explain about salting out method and the calculation. Can I use that equation to calculate the mass of NaCl?
(The equation on the attached file)
Thanks!
Relevant answer
Answer
May i know how much nacl needed to precipitate the collagen solution?
  • asked a question related to Molecular Biological Techniques
Question
6 answers
I  have a stack of 96 well plates containing lysates in RLT Plus. I dont want to process them all at once, rather a few wells per plate to start. Will re-freezing the rest of the plate damage the samples?
Relevant answer
Answer
Ferhat Ozturk, did you really mean RNase? As far as I know, RNase digests RNA and we try to avoid it as hell during the RNA isolation :)
  • asked a question related to Molecular Biological Techniques
Question
9 answers
Does anybody have a lot of experience with Image Studio Lite by LI-COR and quantitative Western blotting?
Relevant answer
Answer
Can anyone share the image studio lite dmg file? I really appreciate it.
  • asked a question related to Molecular Biological Techniques
Question
3 answers
I am using Griess lysis buffer for cell lysis which contains DTPA & EDTA. Will that interfere with CuCl2 when detecting RSNOs in the cells? How to overcome this?
Relevant answer
Answer
Shruti Dumbre hey Shruti, do you have any update regarding this issue?
  • asked a question related to Molecular Biological Techniques
Question
5 answers
Hallo everyone,
I've been having some trouble isolating bacterial RNA from a gram positive organism for a RNA Seq analysis. My problem is that I always get a very intense "cloud band" on the agarose gel around the position where the 5S RNA band should be.. I've tried several protocols and kits, with and without bead beating, Trizol, Lysozyme, but it happens every time.. The first idea was that these are products of degradation, but then again the intensity of the 23S and the 16S bands clearly remains very high. And also, on a Bioanalyzer this 5S band definitely does not look like degradation, but rather as a sharp peak around 127 nt.. Does anyone have any experience with that? If this is in fact the 5S rRNA, why do I get such accumulation, how should I get rid of it and would it temper with my RNA Seq results?
Thank you all in advance!
Relevant answer
Answer
Hello Antony, which protocols have you used? In all of them you get the same result shown in the photo?
  • asked a question related to Molecular Biological Techniques
Question
1 answer
Antibody for NOX4
Relevant answer
Answer
Maybe you can try the NOX4 Antibody from CUSABIO. It had been used to test the HEK-293 cells, HeLa cells, mouse kidney tissue through WB, and get positive results.
For more details, please visit:
  • asked a question related to Molecular Biological Techniques
Question
4 answers
The purpose of denaturation is the break down of dsDNA, so what is the need of intial denaturation while denaturation can do the same.
Relevant answer
Answer
Use of the initial denaturation conditions for all cycles can result in deactivation of the polymerase (depends on type) which is why there is a separate initial denaturation stage for standard PCR conditions. The initial denaturation is sometimes carried out at a higher temperature and for longer than the melting carried out during subsequent cycles. This is because some templates can have a high GC content or secondary structures (like hairpin loops) which inhibit the PCR reaction. By having a hotter/longer initial denaturation it is possible to separate the DNA strands enabling the first round of PCR to take place generating shorter/less complicated products that can then be melted under milder denaturation conditions.
  • asked a question related to Molecular Biological Techniques
Question
7 answers
We would like to dephosphorylate purified proteins (in vitro). How do we decide what type of alkaline phosphatase to purchase?
Relevant answer
Answer
Maybe this Recombinant Human Alkaline phosphatase, placental-like (ALPPL2) works for this situation. You can view the details on this page:
  • asked a question related to Molecular Biological Techniques
Question
14 answers
We are recently trying to isolate RNA of FACS sorted cells with a population of 100K. The cell population we are sorting is very sensitive and we noticed a lot of cell death after the sorting procedure. For RNAisolation we are using RNeasy Mini Kit from Quiagen. Although we tryied to collect the cells with different conditions (PBS, RLT, BSA coated falcon), we did not manage to isolate any RNA. Our unsorted population however gives us a high RNA yield.
Has anybody had similar problems and has a solution we could try? 
Relevant answer
Answer
I have a similar problem! I use same kit and cannot extract good RNA conc (measured in Nanodrop) from FACS sorted cells. I tried to sort in PBS-FBS 2% and then centrifuge my cells and add 350μI RLT Buffer (RNeasy mini kit Qiagen) in the pellet. I usually have approximately 80.000-150.000 cells.
Then I tried to sort directly into lysis buffer, but what about the dilution of lysis buffer when the sample drops in? What is the appropriate amount of lysis to add in what sample volume? Thank you in advance! Jessica Bilstein Anna C Belkina
  • asked a question related to Molecular Biological Techniques
Question
5 answers
I'm in the initial stages of planning a miRNA seq experiment using human cultured cells and decided on TRIzol extraction, Truseq small RNA prep kit, using an illumina HiSeq2500. The illumina webinar suggests 10-20 Million reads for discovery, the QandA support page suggests 2-5M, and I wrote the tech support to ask, who suggested I do up to 100M reads for rare transcripts. Exiqon guide to miRNA discovery manual says there is not really any benefit on going over 5M reads. I was hoping to save money by pooling more samples in a lane, so I was hoping someone with experience might be able to suggest a suitable number of reads.
Relevant answer
Answer
i am working on cardiomyopathy patients Blood samples . and wanted to do miRNA sequencing can some one please suggest how many millions reads i need to sequence 20 millions or 30 millions and also please suggest the platform as well .
  • asked a question related to Molecular Biological Techniques
Question
3 answers
i got mann whitney test value U=1402, i am confused about this so large value. this value is right? i mean if value comes in thousands then no need to worry or there is any problem??? please
Relevant answer
Answer
I want to know the answer too because I got a mann-Whitney of 1754
  • asked a question related to Molecular Biological Techniques
Question
2 answers
I'm going to view localization of certain fluorescent protein in Enterococuccus. As they tend to form chains, and for analysis I need to use single cells images, I wonder if I can fix in first with PFA and then proceed with microscopy? Are they still considered alive?
Another question is if I want to track the appearance of my protein on the cell surface during the cell growth, may I use agarose pad, or it wouldn't divide on it? Can I use BHI agar instead or it will interfere with imaging due to less-transperent properties?
That's where fixing becomes a problem, once fixed will I be able to track division or not?
Relevant answer
Answer
I thought PFA killed everything dead, too, but CD4+ T cells, fixed with PFA, can still activate resting B cells.
Source:
Yellin, M. J., Sippel, K., G Inghirami, Covey, L. R., Lee, J. J., Sinning, J., Clark, E. A., Chess, L., & Lederman, S. (1994). CD40 molecules induce down-modulation and endocytosis of T cell surface T cell-B cell activating molecule/CD40-L. Potential role in regulating helper effector function. The Journal of Immunology152(2), 598–608. https://www.jimmunol.org/content/152/2/598.long
  • asked a question related to Molecular Biological Techniques
Question
10 answers
Hello everyone!
I am using AB Stepone system with Qiagen QuantiNova PCR kit. After doing my first trial, my qPCR results were very strange, the plot are "hook"-shaped (attachment). Anyone know what happen? The machine I use is not calibrated (the last calibration was around 2012~2013), will that cause any problem like this?
Our plate centrifuge are out of order and I cannot centrifuge properly, but no visible droplets remains on the wall. There are still some bubbles on the top liquid surface, is it a possible cause for my results (I thought the bubbles will be removed upon heating)?
I am doing a quantification experiment with 5 standards (300,000 copies, 1:10 diluted to 30 copies). I am new to qPCR and really confused by the result, please help :(
Thank you for your help!!
Relevant answer
Answer
Hi there! I don't know if you solved this already, but just in case I write. I had recently this problem and came to this post to try to find a solution, I found it elsewhere in the end, so I am writing for all the future newcomers as well. The problem with this is that the machine is reading the ROX value on your endogenous control. If you didn't add ROX to your samples (like me), this makes the machine subtract the values of your endogenous to all the samples, resulting in no amplification. Go to "set up" and, on the "assign targets to wells" tag, select the endogenous and select "none" in the reference. Then go to "analysis" and click on the "analyze" button. Magically, all your amplification curves will suddenly appear!
  • asked a question related to Molecular Biological Techniques
Question
1 answer
Hello everyone!
My protein which has a GFP tag is well expressed in nuclei. To visualize cell boundaries in different layers of the root sample, I am planning to counter stain my samples with propidium iodide and DAPI.
I will be taking images using 3i spinning disc confocal microscope.
I want to know if anyone has a working protocol for such settings.
Earlier, I tried Hoechst 33342 to stain nuclei but it appears that even incubating for 30 minutes, the stain was not able go inside the cells as the cell boundaries were clearly visible during microscopy. I tried DAPI as well, but the background was very high even after 3-4 washes and the intensity of nuclei was not great.
I will appreciate if you could share with me your experiences and suggestions regarding my question.
Thanks!
Relevant answer
Answer
Hi, are you planning to use the propidium iodide for both cell boundaries and nuclei?
If your GFP tag is well expressed in the nuclei, you do not need to stain it again. But if the fluorescence signal is not as bright as you want it to be, open the pinhole for more light to reach your sample.
I have used propidium to stain the cell boundary of Arabidopsis roots in the past, and it worked brilliantly. I stained the root for just 5 minutes using the recommended concentration on the bottle.
  • asked a question related to Molecular Biological Techniques
Question
9 answers
Does anybody have experience with ACC (Acetyl Coenzyme A Carboxylase) and pACC detection by western blotting?  It is often used to confirm the status of AMPK activity. The antibodies we use are from Cell Signaling Tech. and they are cited in several publications. However, our anti-ACC does not work at all, while anti-pACC recognizes an unspecific sharp band at 100 kDa and a signal, which  is running around the expected MW 265 kDa,  not as a defined band, but as a “diffused zone”. It should be pACC, because its intensity mirrors that of pAMPK. I am not sure, if it is a problem related to our SDS-PAGE 6% gels or blotting or lysate preparation (RIPA buffer + protease and phosphatase inhibitors, then treated 5 min at 95 °C in highly reducing  conditions (beta-mercaptoethanol, 5% in lysates). We are trying to detect ACC in HepG2 cells and rat liver lysates. This is really frustrating, because according to several publications pACC and ACC should be sharp bands. Please help us to identify the critical problem and get nice pACC/ACC blots.
Relevant answer
Answer
You used 10% gel and you say you had good results letting the dye get to the bottom of the gel.
How many amps did you use for gel run and membrane transfer?
How much time did you spend on these two steps?
  • asked a question related to Molecular Biological Techniques
Question
14 answers
For examination in a laboratory.
Relevant answer
Answer
The most accurate and reliable method for seeing viable Cells is Colony Forming Units (CFU) /ml. Make serial dilution of the desired sample and you can go for pour plating by adding 10-100ul serially diluted sample to petriplate. Incubate for 24 hours and count next day the colonies formed on PDA
  • asked a question related to Molecular Biological Techniques
Question
3 answers
I want to make point mutations/corrections in iPSCs.
Which iPSC vectors would have a high efficiency?
Puromycin selection may be better than GFP/FACS sorting?
Relevant answer
Answer
I do not know how old this discussion is…but…
Actually, doing with RNPs (ribonucleoprotein complexes) of sgRNA and spCa9 protein together with a HDR template using oligos you would get a higher indel efficiency!
It also avoids DNA damage caused by antibiotics usually applied for selection.
So I would give preference to RNPs
  • asked a question related to Molecular Biological Techniques
Question
8 answers
I'm at the moment trying to overexpress a protein known as amidohydrolase from Rhodococcus into e.coli BL21/DE3 using pET28a vector. To confirm the expression, I did western blot with a Anti-6X His tag antibody (given that there is no antibody specifically for amidohydrolase). Surprisingly, my negative control, which is the cells transformed with pET28a without my insert, gives me strong signal at 40KDa. Has anybody encountered a similar situation? Does anyone have an explanation for this?
Relevant answer
Answer
Empty pET28 expresses a 58 aa protein.
Here is the sequence:
MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHisMetAlaSerMetThrGlyGlyGlnGlnMetGlyArgGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEnd
  • asked a question related to Molecular Biological Techniques
Question
1 answer
I had made a cDNA library with infusion smarter cDNA library kit using Stellar Electrocompetent cells, 7 months back. Now when I grow white colonies in LB -Amp they are slow growing and don't achieve requisite culture growth. Also from them I get no plasmid when I proceed.
Relevant answer
Answer
It seems that stellar cell is not compatible to grow in LB broth. I spread recovery medium SOC containing stellar cells after transformation on LB+ carberniciline. They can grow on this plate. However, after 4 months in the fridge, I cannot grow them on LB.
  • asked a question related to Molecular Biological Techniques
Question
1 answer
We are using mouse muscle DNA but ideal sonication condition is elusive. I wonder if anyone used Misonix S 4000 to fragment DNA into 500-1000 bp for CHIP?
Relevant answer
Answer
Hi Zhegnxing, I'm wondering did you figure out the best setting using S-4000 to shear genomic DNA? I'm doing a similar experiment and looking for a relevant protocol.
  • asked a question related to Molecular Biological Techniques
Question
11 answers
Do I need to use DNA ladder or it is not important? what is the programme used for analysisi? and also if I have many samples could I make two electrophoresis gels or they must be in one gel?
Relevant answer
Answer
Dr@aya Gaber..this program offer free trial for one month and there are many videos in their website showing step by step how to analyse gel electrophoresis results
  • asked a question related to Molecular Biological Techniques
Question
4 answers
Hi everyone,
I am trying to improve sustainability in my institute and I realized that there is an overuse of MilliQ water. I believe that for most of the operations done in a microbiology lab (buffers and bacterial media preparation, PCRs, restriction reactions, ligations, DNA assemblies, ...), using distilled water might be sufficient. I was wondering if someone has an overview of which water can be used for which protocol and maybe a list of the operations which really require MilliQ water.
Thank you very much in advance for your help!
Best,
Filippo
Relevant answer
Answer
For molecular reactions (PCR, restriction digests, etc.) it is best to use molecular grade water - purchased in a bottle & not out of a filtration set up.
For buffers & media, regular ddH2O from your filtration system is preferred, but dH2O is good enough.
For washing dishes, use tap water & rinse in dH2O.
  • asked a question related to Molecular Biological Techniques
Question
3 answers
I am having issues with unexpected flourescent bands occurring in my negative control samples (heat inactivated / minus telomerase samples) whilst using the TRAP assay to look for telomerase activity. I have ruled out contamination,  run a temperature gradient for annealing temperatures and varied cycle numbers but this had no effect. I have contacted the assay's producers but they gave no useful suggestions. Has anyone else had the same problem?!
Relevant answer
Answer
Hi, I am having this problem - did you ever figure out how to resolve it? Maybe telomere fragments in the sample?
  • asked a question related to Molecular Biological Techniques
Question
2 answers
Can anyone share a protocol for a ferrozine assay for tissue samples that is reproducible, including the lysis protocol? Also, what is the minimal amount of iron detectable using this protocol and which method was used to quantify the tissue amount (by protein?)?
Relevant answer
Answer
Respected sir,
i want to estimate non heme iron in Fe+2 state, in a protein. Please tell what method i can use? It is very urgent
  • asked a question related to Molecular Biological Techniques
Question
3 answers
I have been working on a TBI mouse model and looking at AQP4. The literature provided by abcam shows the western blot data to have a band at 46 kDa and another one at 20kDa that they cannot identify. My blots have a double band at 48 and 46 kDa and a large band at 20 kDa. Does anyone have any experience working with this antibody? Any suggestions or ideas about what these bands represent?
Relevant answer
Answer
I have been looking into this, as well. I have seen bands at ~90kDa, at ~50kDa, and at ~20kDa. Primary literature points to the ~90kDa band as likely some SDS-insoluble oligomer. But, I can't seem to find anything for the 20kDa band. Have you found anything yet?
  • asked a question related to Molecular Biological Techniques
Question
3 answers
Hello. I am currently developing a protocol for a neutrophil phagocytosis assay using flow cytometry. We will use a GFP-expressing bacteria. Can anyone tell me if the addition of trypan blue will quench the GFP-signal of the non-internalized bacteria? I know trypan blue is used to quench the signal of fluorescently labeled extra-cellular antibodies, but I wasn't sure if it would work if the fluorescent signal was coming from protein inside the bacteria?
Relevant answer
Answer
Did you try to just quench fluorescence of GFP-bacteria (by themselves, no need for neutrophils) with trypan blue?
I would like to try phagocytosis with GFP-yeast, and quenching free yeast.
  • asked a question related to Molecular Biological Techniques
Question
12 answers
I am using Dot Blot study to detect 5hmC level. Dot blot detection is ok but when I use methylene blue stain to check the control, the dots are not so clear. Very faint color I can see on the membrane. Two methylene blue staining methods I used but result is same.
Method-1: Membrane is washed with miliQ water and then treated with 0.04% methylene blue in 0.5M sodium acetate (pH 5.2) for over night.
Method-2: Membrane is washed with miliQ water and then treated with 5% acetic acid solution for 15 min at room temperature. After that treatment with 0.04% methylene blue in 0.5M sodium acetate (pH 5.2) for over night.
Is there anybody who can give some valuable suggestions that could help me to solve this problem?
Relevant answer
Answer
I am putting up a protocol here in case someone is still looking for it. This works quite well with us.
1. Make 0.02% (w/v) methylene blue in 0.3 M sodium acetate (pH 5.5).
2. Stain for 3-5 minutes
3. Destain with water.
4. Change the water several times.
Note: In case you doing this protocol post hybridization wash membrane with 100 % ethanol for 2-3 minutes followed by water and then stain it.
best wishes
  • asked a question related to Molecular Biological Techniques
Question
4 answers
Our group is looking at moving away from traditional western blot methods as there are many more time efficient options now available. I have seen an impressive demonstration of using a few ml of "1-Step™ NBT/BCIP Substrate Solution" to detect protein bands on the membrane. However, I have heard that this technique is limited in that it does not allow for stripping and re-probing with different antibodies. Does anyone have any suggestions as to how this can be overcome? Or are there any other limitations that I should be aware of?
Any advice would be appreciated.
Relevant answer
Answer
Vivek Bhakta Mathema From my experience, it could be a blocking problem when your background appears quicker than your protein band of interest. Try using a fresh blocking buffer. What blocking buffer did you use?
  • asked a question related to Molecular Biological Techniques
Question
6 answers
After rounds of washing and antibody staining, the protein ladder seems to fade. Does anyone know why?
Relevant answer
Answer
This may be an old question but I may have the answer.
There was a time when I first joined a new lab, I adapted to the WB protocol then. Turned out washing with highspeed (say, 110rpm) actually washed away much of my protein ladder. So I moved back to my old washing speed (70rpm) and the ladders were saved, happily ever after
  • asked a question related to Molecular Biological Techniques
Question
5 answers
Hello, I am running an Optiprep iodixanol gradient density to isolate my exosomes. With each run I also have a control which I exact 12 fractions and from the protocol it said I need to calculate the density of each fraction so I will know in which might be my exosomes. 
However, although it might be quite straight forward, I am having difficulties in calculating the density of each fraction. I have been given the coefficient of extinction 320 L g-1 cm-1 and the wavelength 244nm. And with that I dilute each franction to 1:10000 and read the absorbency. However, I do not know how to get to the density from there. Any suggestion?
Thanks
Relevant answer
Answer
@julien siracusa
I have the very same problem. But I have twice higher OD than recommended in all 5,10,20 and 40% fractions. I thought its handling error. So I have repeated again very carefully, no Pipestone error no calculation errors. Yet it's same. If someone knows about this kindly help me to figure out.
Thanks in advance
  • asked a question related to Molecular Biological Techniques
Question
7 answers
I was reading SDS PAGE and methodology it was mentioned, I searched it online but i couldn't find the answer.
Relevant answer
Answer
The ability of Coomassie R-250 dye to bond to proteins is most effective at low pH contributed by acetic acid. Methanol is a fixative which sort of dehydrates the gel. Methanol isn't a requirement. The same concentration of ethanol works just as well and it is safer.
  • asked a question related to Molecular Biological Techniques
Question
6 answers
I ran samples (looking for c.bovis in swabs and tumors) using the same primes/probes, Taqman Master Mix, DEPC H2O that was used last week on samples that worked fine. These new samples that should had been negative tested positive for c. bovis. I replaced all reagents and reran the samples. Still positive. Some samples are negative and the NTC is negative so I do not thing contamination is the cause. I can not think of any other cause for the false positives. Can anyone offer any assistance? Thank You.
I have also included an image of the results for better visual. 
Relevant answer
Answer
@Maria, have you solved the problem with false positives? I have been getting similar false positives only in Hex probe tagged gene in a cheaper enzyme only at Ct between 29 and 34 which is typical for all the false positive cases. In other standard enzymes it does not occur though the primer mix used is the same. This is a multiplex reaction and the other probes are FAM and Rox which are working good. The shapes of the curves for false positive are mostly sigmoidal and some are non-sigmoidal. Please help.
  • asked a question related to Molecular Biological Techniques
Question
6 answers
Hi, 
I'm trying to disrupt some genes of the genome of E. coli. For achieving this, I'm following the protocol of the P1 phage in which this phage transfers genetic material from one strain to another. The recipient strain is from the keio collection, so I don't have any problem with the selection of the colonies which were succesfully trasduced.
But after the confirmation of the disruption with the antibiotic screening and PCR, I tried to remove the Kn resistance using flippase. The flippase is a recombinase that recognize FRT (Flippase recognition target) sites and removes all the flanked area.
This plasmid must be electroporated and then grow it at 30°C, because it has a temperature sensitive ori.
I have done all of this but I have not obtained any transformant. Do you have any clue of why I don't get colonies? Can you give me some tips?
Relevant answer
Answer
Mercedes Vazquez Can I as you what plasmid you are talking about? Is it pCP20?
Many thanks?
  • asked a question related to Molecular Biological Techniques
Question
6 answers
Hello
Today i want to ask about whether there is optimal condition of centrifugation
(speed, time, and temperature) to obtain cell pellet.
I have already searched many protocols, but i only can find those mentioning about bacterial culture.
What i want to ask is about the optimal condition of centrifugation to get pellet of HUVECs. For me, i collect HUVECs to 1.5ml tubes with 1X PBS and I spin down the samples with small centrifuge for about just 30 seconds.
Then i can see the pellet at the bottom.
But i learned that there are numerous conditions of pelleting down cells
so i want to ask whether my protocol described above is ok
Thank you very much!
Relevant answer
Answer
Hello
Using 400g for 5 minutes in tubes of 15 and 50mL I have been able to form pellets of cell lines and tissue cells in suspension without problems. To pellet 96 well plates, I have used 900g for 2 minutes.
Best.
  • asked a question related to Molecular Biological Techniques
Question
7 answers
When determining titer of T7 phage by plaque assay, I get a gradient of plaques where one side of the plate is clearing and the other half of the plate is a lawn of bacteria.  Anyone experience this issue?
Relevant answer
Answer
AS YOU KNOW WELL THE T PHAJES ARE OF TWO TYPES
the ODD. T1, T3, T5 T7, and
THE EVEN ARE T2.T4, T6,
As I KNOW LOT OF WORK AND LITERATURE EXITS BECAUSE THE PLAQUE SIZE ID GOOD ENOUGH TO OBSERVE AND EASY TO WORK AND DETECT MUTATIONS
AND VARIOUS KINDS OF EXPERIMENTs have been done
With odd phages the plaque size is too small and little work has been done
  • asked a question related to Molecular Biological Techniques
Question
4 answers
there are many methods to mtDNA extraction from human blood , what about the positive control used as marker in gel electrophoresis
Relevant answer
Answer
How mtDNaA is extracted from genomic DNA for next generation mtgenomics?
  • asked a question related to Molecular Biological Techniques
Question
3 answers
Every time I try to isolate DNA, it ends with a smear like band on gel, or the pellet obtained is very little. Currently I am using a protocol which requires 2 days to isolate DNA.  If possible please suggest a protocol that enables isolation within the same day.
Relevant answer
Answer
Thank you for nice queries. Total genomic DNA (gDNA) from milk could be extracted using different protocols including automated platform like Maxwell 16 (Promega). See the the latest protocols in: https://doi.org/10.1016/j.ygeno.2020.09.039.
Howver, for RNA extraction, milk should be collected aseptically, and transported to the Laboratory keeping in a RNA later solution. The extraction of RNA from milk somatic cells or else using different cutting-edge protocols. See the protocols in the following manuscript: http://dx.doi.org/10.3168/jds.2016-11184; https://doi.org/10.1007/s40011-017-0955-8.
  • asked a question related to Molecular Biological Techniques
Question
5 answers
Which is the best method and software for Microsatellite markers analysis?
Relevant answer
Answer
NTysys is fine i think
  • asked a question related to Molecular Biological Techniques
Question
7 answers
Urgent for help!!!!!
I want to elute proteins fron Aminophenyl-m7GTP (C10-spacer)-Agarose(Jena Bioscience).
I searched some articles and some answers on Research Gate, there are many different ways. I want to elute it with sample loading buffer,and then running SDS-PAGE gel. 
I want to know how can I wash unbinding protein away from the Agarose? Do I need to check the concentration again after I add same volume of loading buffer to the Agarose? 
Can some tell me detailed procedure to do this?
Thanks advance.
Relevant answer
Answer
I know I'm "bumping" a now ancient thread, but I recently used these beads (Jena, AC-155S) to successfully test binding of a plant 4EHP-homolog to m7GTP. Elution was performed by resuspending the beads in 1x Laemmli sample buffer and boiling at 95C for 5 min. A few observations that may be helpful for people considering using these beads:
  • After samples cool, the agarose will congeal as a mass at the bottom of the tube.
  • Using a 4:1 elution buffer to bead ratio seems to result in only half the initial volume of elution buffer as pipettable; the other half gets incorporated into the congealed agarose. 7:1 elution buffer to bead ratio seems to work well.
  • I wonder if elution issues observed by others is due to proteins getting trapped in congealed agarose?
  • asked a question related to Molecular Biological Techniques
Question
6 answers
Hello everyone,
I am trying to clone 4 inserts (816 bp, 864 bp, 1226 bp and 1699 bp) into Xho I digested pBSK (+) vector (2598 bp) using NEB's Gibson assembly kit. All the fragments used are gel purified and have a good yield. I have set up reactions by taking 0.025 pmoles of vector and 0.05 pmoles of each of the inserts, with a total reaction volume of 15ul. The reaction is incubated as per the manufacturer's instructions. I have been checking my assembly on the gel before I transform it into XL-1 Blue competent cells and I observe a faint band of expected size (7.2 kb). However, I am unable to get any recombinants post-transformation and end up with only self-ligated vector. The vector has been XhoI digested, gel extracted and treated with antartic phosphatase to prevent self-ligation. Can someone suggest tips to drive the reaction so as to achieve more efficient assembly of the fragments?
Thanks a lot!
Kajal
Relevant answer
Answer
follow these points and you will most likely get cloned vector:
1) Keep the molar ratio of vector to insert as 1:3. Your insert DNA should be 3X fold and not less than 3X.
2) Insert should have complementary bases (on 3' and 5' ends) to your BB of at least 20-25bp.
3) Strictly try to use the vector BB as a PCR amplified product and not as restriction enzyme digested product. Most of the time, there is little amount of undigested vectors in digestion reaction and this causes the false colony appearance on ampicillin plates.
Hope this helps.
  • asked a question related to Molecular Biological Techniques
Question
6 answers
From the standard text, I found that ethidium bromide intercalates the double stranded DNA, then how single stranded DNA can be visualized by ethidium bromide? I am curious about different visualization methods and mechanism?
Relevant answer
Answer
I agree with Mohsen Ashrafi . In addition , Ethidium Bromide is carcinogenic dye and there are another dye more safety like SYBER GREEN.
  • asked a question related to Molecular Biological Techniques
Question
1 answer
If I vortex stock primer for PCR strongly, will it break easily?
Relevant answer
Answer
Don't vortex too much. But you can vortex gently for 5-10 for proper mixing. It would not break primers.
  • asked a question related to Molecular Biological Techniques
Question
7 answers
I'm trying to find the best way to measure acetylcholine (ACh) levels in brain areas of interest. I've used some commercially available kits but the results are not great (maybe problems with detection of ACh).
Relevant answer
Answer
I tried the kit few times and it gives higher values for Free choline than Total choline hence negative values for acetylcholine. Even if all acetylcholine is converted to choline during brain tissue processing, free choline should never exceed total choline. Is it possible that choline acetyltransferase is rapidly converting choline to acetylcholine?
  • asked a question related to Molecular Biological Techniques
Question
10 answers
I need to know if you can induce gene expression by injecting it into the mouse. There are plenty of examples of using the system in cell culture but I wasn't able to find anything about using it in animals. Thank you.
Relevant answer
Answer
At least in zebrafish embryo, cumate is too toxic to be used. Even 1xconcentration of cumate (from SBI) drove all embryonic death within few hours. So, I do not recommend cumate applying to in vivo experiments. Eric R Hugo
  • asked a question related to Molecular Biological Techniques
Question
10 answers
Hii
I need help. I need to send some RNA samples to my service provider in US. I am planning to send my samples in Ethanol and Sodium acetate precipitate form. But my issue is I dont have an option to send in DRY ice or cold condition. Can anyone tell me will my RNA in ethanol and sodium acetate be stable at room temperature? 
I know, until there is no RNase in my samples, nothing can happen. As we all tried till now is keeping in -80C until use, But still anyone tried to keep the RNA in ethanol and sodium acetate at room temperature for more than 24hr? I know i may be asking a  stupid question, but accidents can happen always in someones life. Like, forgetting to keep the samples in -80C, something like that happened to anyone?
I need answers from experienced hands so that with confidence i can send my samples in room temperature in ethanol and sodium acetate. 
Relevant answer
Answer
Hi Imene,
Happy to help you. I isolated total RNA through the traditional method (refer to my publication for protocol) and checked the quality. Samples were shipped in normal Ice by adding 2.5 volumes of absolute ethanol. One of my friends carried it in luggage while flying and RNA was stable. The samples had around 7 RIN values after reprecipitating and got good RNA seq results. For the safer side, you can follow the kit method (Qiagen or Sigma) so that it will be more stable and can avoid RNase contamination by handing the traditional method. Keep in mind, if you handle the samples properly and perform a good RNA preparation, they will be stable. Even you can isolate and try to keep it in ice for 1 or 2 days and have a check before sending it. I don't know anything about your samples (plant or animal) also how the properties of the tissue. If you can share the information, I will be happy to help you more.
Good Luck!
Best,
Sajeevan
  • asked a question related to Molecular Biological Techniques
Question
4 answers
I have been trying to arrest immortalized MEFs using double thymidine chase, I have used two different protocols, in one I added 2mM thymidine for 15 hours released 10 hours and back to thymidine for 17 hours, but when I took the cells to flow cytometry I cannot see a nice peak at the beginning of S phase instead I saw an increase of cells on S phase indeed but I still can see a G1 peak and some cells on G2/M, has anyone arrested these cells or has any suggestion? Thanks,
Relevant answer
Answer
I have not tried Thymidine block yet and I am not getting nice results with Nocodazole. The method that has given me a reliable result to track S-phase is serum depletion.
12h with depletion media (DMEM+glutamine+0.1%FCS) and then release in DMEM+glutamine+10%FCS.
I hope it helps.
Edd R-C
  • asked a question related to Molecular Biological Techniques
Question
3 answers
Is there any DAB staining method for ROS detection in pea leaves??
Relevant answer
Answer
We only use in situ detection of H2O2. Histochemical staining is described in:
  • asked a question related to Molecular Biological Techniques
Question
8 answers
The storage buffer of the RiboLock RNase reagent contains 50% (v/v) glycerol that prevents the overdrying of RNA. My question is what if we would use ethanol along with glycerol for DNA drying too.
Тhank you.
Relevant answer
Answer
Follow
  • asked a question related to Molecular Biological Techniques
Question
9 answers
I had done PCR in two batches. I had casted a agarose gel using two combs. I had loaded few samples from the first batch (most of them had been already visualized and had given good amplification) and then ladder and the few samples from second batch in the upper well. The remaining samples of second batch were loaded in lower wells of same gel.
After visualizing the gel under UV no bands were seen in the upper wells except the ladder while good amplification was seen in lower wells.
Why it occurred?
Relevant answer
Answer
The same thing I experienced today. I am sure, I have loaded the samples with dye, and at the upper left lane, I used the ladder. So it happened!
  • asked a question related to Molecular Biological Techniques
Question
6 answers
Dear all, I am currently using Qiagen miRNA profiler plates (catalogue number: MIBT-659ZF-24) to identify deregulated miRNAs in cattle under certain physiological condition.