- Beston F Nore added an answer:9Does anyone know the sequence for the multiple cloning site in Lonza's pmaxGFP or at least the order of restriction sites upstream of GFP?
Lonza provides a plasmid map of pmaxGFP showing the KpnI, NheI, Eco47III, and AgeI restriction sites upstream of max GFP and BglII, XhoI, and SacI downstream. Does anyone know if there are additional sites upstream of GFP? I would like to try and clone my GOI from another expression plasmid into this one due to its small size.
Attached a map of the MCS sites ? I hope that cover the answer.Following
- Prachi Singh added an answer:3MeDip Sequencing can be performed with little sheared gDNA?
I have isolated gDNA using Quiagen kit but the quality of DNA on gel was not up to the mark( picture attached). Can I send these samples for MeDip sequencing as for sequencing anyways they have to sonicate the dna so if degradation is there that should not affect my sequencing results.
Thanks and regards :)
I have used quiagen kit and isolated the genomic dna ..it was not a mannual isolation so i think purity is not a matter...
thanks sir for ur responseFollowing
- Uwe Borgmeyer added an answer:2How can I select the cell that are gene-edidted by CRISPR/CAS9 system?
I want to know that If I transfect a human cell line using transcriped sgRNA, CAS9 protein, and a homologous gene
.How can I select the edited cell by the homologous gene ?
How can I use antibitic marker to select?
or is it not needed to use antibiotic marker to select ?
I recommend to read the guide from Addgene. It will be helpful in designing an experiment and explain the possibilities to isolate edited cells:
- Byung Kwon Jung asked a question:NewAnybody know detail of Red recombination technics?
these days, I'm doing gene knockout mutation to use Red recombination including pKD119 (having araBAD promoter, exonuclease gene) and pKD3 (TetR) as helper and template (cassette) plasmid, respectively.
For mutatagenesis experiment, first, pKD119 transfer to bacteria (Serratia fonticola) ,then, transformant grow at 30 degree until OD600=0.3.
And, L-arabinose (final concentration=0.35%) add to culture broth and exonuclease induced at 37 degree for 45 min.
Following to homology casstte consist of chloramphenicol resistance gene flanked by 36 bp target gene transfer to S. fonticola having pKD119 plasmid by electroporation (1350 V or 2500 V, 1mm or 2mm cuvette).
After electroporation, bacterial cell recover at 37 degree for 3 hr, maybe this process is recombination step.
Then, recovered cell spread onto LB-chloramphenicol agar and incubate at 37 degree.
But I could not fount colony.. what's the problem?
this protocol refer to gene bridge company and other research paper which mentioned E. coli, Serratia, Yersinia, Salmonella species is possible mutaion to use Red recombination system.
My S. fonticola is not only belonging to Serratia, but also close to Yersinia.
which bacteria species classified as Enterobacteraceae family.
My Cassette concentration is enough (200 ng/uL) and exactly amplified.
And, helper plasmid pKD119 is successfully replication in my bacteria, I checked.
What is the problem in my expriments..
Chloramphenicol concentration is high? or another problems?
Please tell me solution.Following
- Daniel Dehany Scott added an answer:2In autosomal and X-linked dominant mutations, how does the mutated gene override the normal allele, which can still produce a normal gene product?
In individuals who have a dominant mutation on one gene, and heterogenously a working copy of that gene on the other partner chromosome, how does the mutated allele override the normal one which can still produce a normal gene product?
Moritz has given a very good answer that covers many of the mechanisms. I just wanted to add a few addenda.
First, it's possible that the mutated protein may interfere with the function of another pathway different to its own, or may become pathogenic - for example, if it aberrantly interacts with another protein, is mislocalised to a different sub cellular region, or if it forms pathogenic aggregates, amyloid or other structures (as in amyloid diseases, or trinucleotise expansions like Huntingtons). While this would technically be a subset of "gain-of-function" mutations, it's a little different as they're gaining new, non-physiological functions rather than increases in their normal functions.
the other thing to keep in mind specifically for X-linked autosomal diseases is that, in female cells specifically, one X chromosome is always inactivated, meaning that cells will only express genes from one copy of the X chromosome. Different cells may inactivate different copies of the chromosome, so you can get mosaicism for the expression of a normal or pathogenic allele from a heterozygous X-linked locus. In this case, the particular tissues/cells expressing the different alleles will also affect the pathology of the disease.
hope that helps,
- Anja Hühnlein added an answer:1QPCR plate set up help?
I am just learning QPCR and have a plate set up question.
I have 5 bacteria strains with a treated and a control sample for each. I will also have 4 genes to look at the expression levels of. Since I am doing SYBR green, I need to run normalizer genes as well for each sample.
I am a bit confused on how to set the plate up. According to the manual:
EXAMPLE - STRAIN A
I will have Strain A treated sample and STRAIN A control sample for my first GOI (GOI#1). I will aslo have to run my normalizer gene (to make sure the treatments don't effect expression levels - 16SRNA) for each sample as well. If I do duplicates (technical reps), that is 2+2+2+2 (x3) as I have 3 biological replicates. THis is 24 wells.
I can fit 3 strains (24 x 3) on a 96 well plate if I am looking GOI#1. I need to have some controls (NTC, No RT controls).
My question is I still have 2 more strains to look at. Can I just start another plate with those strains and carry on like I did for the first plate or do I have to LINK the plates somehow?
Then I have to do that for each of my GOIs. I want to make sure I have the setup right.
If anyone has any advice, that would be great.
You can link your plates by pipeting an interplate calibrator. The IC can consist of diluted PCR product of the target DNA or cDNA sequence. See http://www.tataa.com/products-page/quality-assessment/tataa-interplate-calibrator/Following
- Rogério de Jesus added an answer:9What is the best DNA extraction kit for a large amount of tissue?
What is the best DNA extraction kit for a large amount of tissue (approximately 500 - 800 mg)? The tissue may not be fragmented into multiple samples for analysis.
Javidfar, the tissue is entire brain of mouse.
Which protocol of Qiagen kit do you use for that amount of tissue?
Behnam Javidfar and Huda Hammed, the tissue is entire brain of mouse.
Which protocol of Qiagen kit do you use for that amount of tissue?Following
- Peter Eck added an answer:2Does an alternative method exist for measuring riboflavin uptake without using radiolabbeling?
I need to study the uptake of riboflavin in some cell models. I was wondering whether anyone can suggest me a way to measure the level of uptake riboflavin without using radiolabeled riboflavin....
In all the literature that I looked at, scientists have been using tritium-riboflavin [3H]-riboflavin.... does anyone have experience with other isotopes such as C14 that may work in the same way?
if you try to avoid radiolabeled compounds, measure the compound itself. there should be plenty of methodology out there, e.g. HPLC with different detection methods, e.g. MS (Mass spectrometry).Following
- Joanne Kamens added an answer:7Any advice with my restriction digestion: is the xbaI site no longer available after stop codon?
I recently found a phenomenon that I was working on genetic engineering. the plasmid vector was cut with notI and xbaI, and the insert segment PCR is also flanked by these two sites. but there is a TGA right in front of 3' xbaI. so the insert is in this format:
genetic engineering was successful and positive colonies confirmed. However after this, I found the xbaI site is no longer cutable. even weird is that, my sequence reading software stops detecting the xbaI site, even tho- the xbaI sequence is right there. (and you can try put TGATCTAGA into your own sequence software it probably wont recognize xbaI)
I wonder if the TGA somehow disrupted the xbaI cutting ability, or any other property I havent heard about xbaI that causes this?
To learn about the correct strains for different types of plasmids check out this Plasmids 101 blog http://blog.addgene.org/plasmids-101-common-lab-e-coli-strains
(or download the free Plasmids 101 ebook - if you are going to use plasmids, may as well know a lot about how they work!)Following
- Najim Ameziane added an answer:6How to study the change in gene expression in control and knock outs?
Iam starting a new project in which Iam silencing down two genes by lentivirus. Lets say gene x and Y. I have two single and one double knock downs. I want to study the expression of the genes involved in cancer progression, for this I am planning to do RNA sequencing for the single and double knockdown.
My question is, if I do RNA seq, will I be able to compare the gene expression levels of say MAPK pathway in the control and the knockdowns. If Iam not right, please suggest me a method, were I can be able to study the genes involved in cancer progression (control Vs knockdowns).
If you're solely interested in the involvement of a certain pathway (e.i. MAPK), then a targeted approach using specific qPCR primers for components of that pathway is the way to go. However, if you want to take an unbiased approach and investigate differential expression between the different isogenic cell lines then RNAseq would yield much richer data. When performing RNAseq consider replicates to obtain statistical significance. There are freely available tools for non-bioinformatician with corresponding tutorials you could use. Seqmonq and Altanalyze are tow examples. Finaly, it's always advisable to discuss with a bioinformatician for help with design of the experimental procedure.
- Nisha Rathor added an answer:7Is a non denaturing agarose gel a good method for visualizing RNA integrity?
As my experiment involves RNA isolation, I just want to quickly check the total RNA integrity on a gel and confirm it on a UV-spectrophotometer. For that purpose, is a non denaturing agarose gel of 1-1.2% in TBE buffer a good option? Also a final volume of 2ul ethidium bromide is sufficient or should I add more for viewing RNA? I have seen from other sources that ethidium bromide amount is added more than usual unlike what is used for DNA. And is EtBr a good chemical to visualize RNA under UV? Kindly input your valuable suggestions.
PS: I am planning to incubate the RNA samples at 70C waterbath for 3 mins and place them on ice before loading on to the gel. Also my lab does not have an Agilent Bioanalyzer which I am sure is the best solution for RNA quality analysis but please suggest some possible solutions for agarose based method as well.
u can use non-denaturing agarose gels, i would like to suggest, make your buffer in DEPC treated water, and run the gel at 150V for 10 min. As RNA is very unstable, visualize the gel quickly after runFollowing
- Sebastian Alonso Quezada added an answer:3In an In Situ Hybridization, how can someone tell that a probe is labeling a certain gene and ONLY that particular gene?
I know the probes are specific, but as in PCR, sometimes you might be annealing with something else, specially if you designed the probe yourself and its specificity hasn't been validated.
How do yo 'validate' such a probe beforehand?
Thanks for the replies =)
Simone, I'm at the moment just designing probes and thinking of the best way to address my questions through ISH, and that reply of yours actually helped me a lot along with Chellakkan's. I need to find some suitable tissues for positive and negative controls for my probes.
Thank you very much again =)!Following
- Michael J. Benedik added an answer:13What do you think is the best system to do gene knockout in bacteria genome?Many options are available. From your experience, what choice is the most efficient tool for gene knockout in bacteria, considering the time consumption, robustness of protocols, availability of reagents and plasmids.
Daniel - there is a pretty vast literature on generating gene knockouts in various Vibrio strains. I would review the literature for the strain that you are using (different Vibrio's are quite different and may not be generalizable).Following
- Jack M Gallup added an answer:7Primers are not yielding bands, could they be working?
I have designed qPCR primers for multiple genes, tested them using regular PCR (35 cycle) and agarose gel and most of them are yielding a band of the expected size, However, some are not giving any band of any size which indicate they might not be working for some reason but a friend suggested that the primers could be actually working but the band is not sharp enough to appear on the gel and that I should test them in a qPCR reaction since SYBR green is more sensitive than the Taq poly in detecting any amplification. Is that true? I don't want to waste my time if this is not correct.
Dr. Renee Horner of Ambion has reported using primers demonstrating as low as 48% efficiency in qPCR. As long as the apparently stable (albeit low) efficiency is acknowledged in the ensuing mathematics, it can be useful.
Efficiency itself (in the PCR/qPCR) sense is a fairly misused idea. The only reason efficiency ranges like "90-100%" or "80 to 115%" and so on have been cited is due to lack of precision and/or the existing potential caveats in the technique itself.
As we all know, efficiencies above 100% in a '2 to the n' process is molecularly impossible. Linus Pauling once proposed a triple helix for the structure of DNA - in which case a '3 to the n' process would have been the playing field if he was correct.
So, reaction inhibition (either introduced by carryover contaminants in the sample preparations, or by too much target template being added to the reactions) of the first point or points in the dilution curves, and/or flattening of the curve at the dilute end by primer dimer and/or non-specific product formations/signal contributions have haunted the PCR/qPCR technique since its inception - giving rise to the mythic lore of efficiencies exceeding 100%. Efficiencies exceeding 100% always indicate something that needs to be addressed and fixed.
Perhaps just as troubling are efficiencies below 100% - yet, interestingly, these efficiencies seem quite stable and consistent (R2 values for dilution curves often >0.97) - indicating that the suboptimal kinetic certain sequence interactions attain by PCR is an intrinsic property of the PCR gymnastic itself when faced with the Tm and thermodynamic (known and unknown) properties of the specific primers and templates involved (all coupled with the effects of diminishing reactants, accumulating product and possible fluor accumulation over the course of 35 to 45 cycles).
Efficiency is not even constant from cycle to cycle (as demonstrated eloquently by Rutledge and Stewart in 2008 and elsewhere).
When (if) low efficiency is a stable phenomena, and the standards and samples have the same matrix interference to the point that both can be considered 'equally deleterious' to the efficacy of the PCR/qPCR, then using calculations that correct for efficiency is an approach that can be helpful. In the end, it is always best to seek to design primers that exhibit ~100% efficiency, 100% of the time, but perhaps this is not the planet on which to expect that to happen 100% of the time. I would say: 70-100% efficiency is a better/more realistic range if indeed dilution curve (or dilution profile) stability is observed (high R2) therein.
Once you find yourself dealing with efficiencies above 100%, you are either adding too much sample to the reactions, and/or you have non-specific product formation(s) and/or primer dimer contributions to signal; all problems that need to be eliminated or creatively avoided before expecting good information out of the endeavor. Efficiency may actually be a "ghost' of some sort, a specter of highly-assembled reactants in a tube - which is a scary thought. When I found out that rainbows weren't actually created by a magical flying elf with a multi-colored paint-brush, it changed my view of the world ;]. Keep a healthy suspicion about the notion of PCR/qPCR efficiency - even when working at apparently 100%, because the very process by which you calculated 100% efficiency from a dilution curve, isn't even 100% efficient from cycle to cycle itself... therein lies the rub.Following
- Debaprasad Koner added an answer:5Can someone help me with Real time PCR of genes at acidic pH?
Hi All, i am working real time PCR of certain genes at acidic pH. i have done the same experiment 4-5 times one year back and i got upregulation of certain genes. This work is also published by other groups. Now the difficulty is that i am unable to reproduce the data. I have tried changing everything, ranging from strain, and treatment conditions, SYBR green,CDNA synthesis kit, DNase treatment. I feel that my treatment is somehow not working since positive controls are not working fine, i.e. genes which show upregulation at acidic pH are showing downregulation. I also tried checking for RNA degradation, DNA contamination etc but everything seems fine.I dont understand what is happening?
BTW i work on tuberculosis and i am following exactly same protocols which i used earlier, but no troubleshooting seem to work for me.
It might sound stupid, but is it possible that milliQ water that i am using has some issues?
I also faced a stupid issue (wrt cDNA). I diluted cDNA to 1:5, 1:10 and 1:50 (made from 500ng RNA) and did RT-PCR so that Ct values fall between 15-28. But strangely, all the cDNA concentrations show same, i mean exactly same Ct value. So now i feel that cDNA is the issue. How to make sure if my cDNA is fine.
How much difference are you getting between two(previous and present) experiment? Is it significantly different? Are you using same endogenous control like before? Try to maintain same sample parameter like previous experiment. Pipetting might be an issue. Kindly try with calibrated pipette.
Hope for the best.Following
- Katherine Balasingham added an answer:7No amplification for 400bp, but can easily get amplification for 700+bp?
Bit of a puzzling scenario here for some samples...
DNA samples extracted from water using BioBasic soil kits, will amplify 16S rRNA(~400bp), and ~700bp region of COI gene very well.
However, the same samples will not amplify COI regions of 300-400bp, which is my target.
PCR negatives and positives work all the time. Tried temperature gradient, new reagents, diluting the samples (1:3, 1:10 dilutions), loading 2.0ul of template (typically load 1.0 or 0.5 and both don't work) and double PCR but no results for that size. I have had issues with inhibition before but wouldn't inhibition affect both primers?
Why will it not amplify a shorter target, but will work for a larger region?
These primers are species specific and work well at diff template concentrations. I have designed a few sets that work on controls..just not the eDNA samples.Following
- Michael J. Benedik added an answer:3How can i get Image Lab™ Software #1709690?
we need Image Lab™ Software #1709690 for bio rad to visualize chemiluminescence, the original software actually got corrupted and getting from company is quite costly any other way to get it?
If you have the license for the software, then BioRad should allow you to download another copy at no cost.Following
- Andrii I Rozhok added an answer:4How can different DNA templates affect annealing temperature?
I ran an optimization of PCR condition using a pair of degenerate primer for two genomic DNA as samples. Sample A was extracted from Gram negative bacterium whether Sample B was extracted from Gram positive bacterium. Sample A got a higher GC content for its genomic DNA in comparison to Sample B. The optimization results for the same degenerate primer showed that annealing temperature for Sample B is higher than Sample A.
Can anybody help to explain this phenomena?
I think, first of all, that the general principle is the same for both exact and degenerate primers - the template sequence has a direct effect on the annealing temperature. Given that the primers used were degenerate, meaning that they were a mix of slightly varied primers, differences in the template sequence affect the proportion of primers that work with various success under various temperatures. Then when you calculate the optimal temperature, it will certainly vary based on the template, as basically you get different "effective" concentrations of the primers that worked among those in the mixFollowing
- Zongyou Guo added an answer:2Anybody tried a CRISPR_based gene activation system?
I am curious how this system:
lenti sgRNA(MS2)_zeo backbone
Is this a good system?
Thanks.Thanks for the info. I am going to try it out and see how everything goes.Following
- Dong-Jiunn Jeffery Truong Zhang added an answer:2Did anyone try the lentivirus for CRISPR and nCas9 of abm?
I want to knockout a gen and I would like to know how to select the clones without the protein. There is a antibiotic sistem in the plasmid but that does not provide that the mutation take place in every cell if I am using a pool of them. Thank you!!
If your GOI is expressed in your target cell line already (because some people first modify one easy to transfect/harvest cell type and afterwards transdifferentiate it to another), you can create a "Donor" sequence like this:
5'-HA_SA_P2A_Marker_2xpA_3'-HA when targeting intronic sequence with already at least one coding exon before.
5'-HA_SA_Marker_2xpA_3'-HA when targeting 5'-UTR (Don't forget start codon for Marker)
5'-HA_P2A_Marker_2xpA_3'-HA when targeting exonic coding sequences
HA: Homology Arms
SA: Splice Acceptor
P2A: Porcine Teschoivirus 2A peptide
Marker: Can also be fluorecent proteins (especially when you have FACS sorting systems in your facility) and not only selection markers. I prefer using fluorescent proteins since selection often leads to multiple non-specific integrations especially when you use too much antibiotics.
2xpA: 2x strong pA signal to really terminate the transcript at this point (e.g. SV40 late pA + bGH pA), because you might have very few times a read-through
Pay attention that all is in-frame which is important when using SA and P2A sequences. Also be aware of isoforms and alternative TSS.
I prefer to method targeting an intron inlcuding loxP sites flanking the "SA_P2A_Marker_2xpA" region because then you can show elegantly that you can rescue the phenotype or whatever by cutting this "stop-cassette" out with Cre and showing the reviewers that your actually phenotype is because of your knoch-in...Following
- Shilpi Nagpal added an answer:9Has anyone tried removing some part of a gene from the center of the gene using overlap PCR?
The gene is cloned in a vector and we want to remove some part of gene from its center using overlap PCR ( approx 50 bases needs to be removed).
Size of gene is 2.7 kb and size of vector is 5.5 in which this gene is cloned.Hi Anant, I always did Gel extraction of each fragment after PCR amplification. And then this gel extracted fragments were used to make overlap product. I have sequenced these overlap products as well, and they show correct sequence , with correct cloning sites and extra nts after cloning sites. But when I used this overlap PCR product for further PCR amplification, I did not get any product. Rather I got smear. Also, I am using this overlap product which I get after stitching upstream and downstream part for cloning.Following
- Spiros Vlahopoulos added an answer:1How long is human Interleukin 8 gene promoter? Is there any distant regulatory element binding site?
Now I am searching for the information about human interleukin 8 gene promoter. I am wondering, although the -1 ~ - 500 region is enriched with regulatory element binding sites, what is the full length of the IL8 promoter? Is there any distant regulatory element binding site, like 2kb prior to the start site? Thanks for the help : )
Indeed the proximal promoter has given a lot of information on the inducibility of the il8 gene. However, there is no limit in the distance of DNA sequences that can contribute to changes in the expression of a gene.Following
- Syed A Ali added an answer:3How can I target an intrinsically disordered protein, secreted from a pathogenic bacterium, in the cytoplasm of an eukaryotic cell?
You can molecularly engineer your protein (I assume your anti toxin is a protein) so that on N-terminal it contains a signal peptide and on C-terminal it contains a cell penetrating peptide (CPP). The signal peptide will assist with secretion whereas cell penetrating peptide will fascilitate its internalization into the target cells. You may want to test various signal peptides and CPPs to come up with optimal combination. Finally you get the entire cassette integrated into the genome of producer bacteria for stable expression.Following
- Jai Ghosh added an answer:7How can I identify the number of genes from cloned cDNA sequence?
I am trying to determine the copy number of a gene which control flower development in B napus. I have done the following works to do that.
1. Isolated mRNA from from B napus flower bud and converted to cDNA.
2. Did RT-PCR using primer situated on the 5' and 3' UTR (based on genome sequence) and cloned the RT-PCR product in pGEM T Easy vector.
3. Miniprep 4 cDNA clone and sequenced it. In the sequenced file I have seen a number single nucleotide difference among these clone.
Now I am not sure whether they are different genes or different allele in the same locus. How can I analyze my cDNA sequence data whether they are different genes or different allele. Also what further experiment I can do to confirm the copy number of this gene.
You can, you carry out DNA amplification and then sequence it in a DNA sequencer.Following
- Joko Logis added an answer:11Problems with colony PCR of Pichia pastoris - any thoughts?I need a protocol of colony PCR for confirmation of inserts after the Pichia pastoris electroporation. I performed a protocol, however, it didn't amplified any bands. So, I would like to compare to another one. Does somebody has any protocol (from collect colony until the pcr mix) for colony PCR of P. pastoris transformants?
Hi Fernanda, would u mind sharing the protocol for method number 3? :)Following
- Joginder Sharma added an answer:25How do I concentrate the PCR products ?
I have PCR product for sequencing and its too low concentration. How do I get more yield of PCR products.
Just rePCR your PCR product. or do the nested PCR.Following
- Paschalis Thomas Doulias added an answer:1Is there any possibility that mice with increased B-oxidation, can still develop fatty liver disease upon aging?
I am studying the mechanism of a protein localized in the mitochondria of liver in a mice knocked out for my specific protein. The liver has increased lipid droplets in aged mice. Also my protein was previously shown to involve in positive lipid metabolism. In this context, to understand its mechanism followed by other experiments, I hypothesize that B Oxidation could be increased in those mice. However, I am not sure if it fit with the observation that those mice also develop fatty liver upon aging?
Is it possible and making sense. Or my reasoning is wrong?
Any suggestion, criticism will be really helpful
I would like to know more details before I give you an answer. Lipids in the liver have 4 different fates, 1. synthesis, 2. storage as TGs, 3. export and 4. catabolism through b-oxidation. The fact that mice accumulate progressively fat may speak for one or more of the above mentioned factors. From one hand b-oxidation may increase but if uptake, or synthesis increase as well the net result is going to be accumulation of fat droplets. What is the protein of interest?Following
About Genetic Engineering
Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc.