- Hui-jie Zhai added an answer:2Which pedigree software do you suggest ?
We are looking to buy new pedigree software for our lab. I have used cyrillic, I found it difficult to convert it to JPEG.
export your pics into PDF files, then edit pdf with photoshop, and at last save the pics into JEPGFollowing
- Julie E Gray added an answer:4How many genetically engineered crops have been commercialized?
Genetically engineered crops are being highlighted. How many of them have been commercially successful.
tomatoes, soya, cotton, maize, papaya, oil seed rape, potatoes...Following
- Anton Beletskii added an answer:2Does anyone have a protocol to label DNA with Terminal Deoxynucleotidyl Transferase?
I need to incorporate FITC-dUTP to my DNA sample in a different way than Nick translation or PCR. Does anyone have experience with this enzyme and, better yet a protocol?.
Agree with Sambrook recommendation. For online protocol, look up DIG terminal labeling protocols on Roche/Boehringer Manheim website, they will work on FITC-labeled nucleotides just fine.Following
- Lubica Piknova added an answer:22How 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.
Another possibility is to make another PCR with products of the first PCR. You´ll get more of the original product in the second PCR. It is something like "nested" PCR.Following
- Tamer Mahfouz added an answer:2I’m planning to investigate Red Palm Weevils on date palm and I want Agrobacterium tumefaciens carrying Bt-Cry3Aa gene, could any one help me?
I’m planning to investigate Red Palm Weevils on date palm and I want Agrobacterium tumefaciens carrying Bt-Cry3Aa gene, could any one help me?g
Dear DR.Yuan-Yeu Yau
I hope that you are well
Yes i requesting Bt-Cry3Aa gene. Where i can found it.
- Ndeye Khady Thiombane added an answer:4Would only one nucleotide polymorphism between 2 alleles be enough for the CRISPR guide RNA to target one allele and not the other?
I would like to use CRISPR to introduce a heterozygous point mutation. Would that be possible given that there is only one nucleotide difference between the 2 alleles at the site of interest where I want my guide RNA to bind.
Great, Thank you Albert!Following
- Michael J. Benedik added an answer:5Has anyone tried DNA ligation for long fragments (7kb+11kb)? Does anyone have any suggestions?
For past one month, I have been struggling with a ligation reaction. I have been trying to insert a ~7kb DNA fragment into ~11kb plasmid and both them have been preparation by restriction enzyme (NruⅠ-HF and ASCⅠ from NEB ). Then the vector and insert have been ligated by T4 DNA ligase with PEG 4000 under 22℃ for one hour. The vector has been dephosphorylated well. The molar ratio of the vector and insert is 1:3. However, I got a unexpected result that the recombinants are only 4kb. I could not understand the result hitherto. Thus far I have not been able to obtain a clone consisting of the 11 kb fragment and the 7kb fragment. Does anyone have any suggestions?
PCR cleanup removes small molecules but not necessarily template DNA. You could try a brief DpnI digestion of the PCR product before ligation or gel purify the PCR product.Following
- Shah Rashid added an answer:4How can we prepare a culturing room for thermophillic bacteria?
How can we prepare a culturing room for thermophillic bacteria?
If you need agitation/shake, then you use temperature control shaker. Otherwise, you can use the laboratory oven.Following
- Edward Alain Pajarillo added an answer:6Why do I get plasmid DNA transformants smaller than my original pDNA vector?
So I always have this weird results during my gene cloning experiments. I perform restriction enzyme digestion (double digest) for my plasmid vector (7.0 kb) and insert gene (1.5 kb). I perform ligation using T4 DNA ligase, incubate for 16 h at 16 C, then E. coli DH5a transformation with proper controls.
After picking colonies, plasmid miniprep, I get pDNA transformants with (circular) size smaller than the control (so many of them). I always get this kind of results even in the past, I can't get proper transformants and one of the problem is this and the other is low transformation efficiency. I am not sure what is the problem? Can anyone help me?
To me it's a host problem, when I switch to E. coli HST08 from DH5a, I get better results and higher rate of successful transformants. But some colleagues said my DNA concentration is too low, although I've been using 150-200 ng of DNA vector and the 5:1, 10:1 or more insert:vector molar ratio even in the past. Any one with the same problem?
Hi Dr. Alfonso
I want to believe that you cut your minipreps with a restriction enzyme to get the lineal form of the DNA? sometimes you can have several forms of supercoiled plasmid DNA that run longer than normal... and if you used different strains they can have different topoisomerases
- I make sure I get 1 cut, by (1) calculating the right amount of DNA weight to be used for digestion. Usually, 1 ug per unit of enzyme for 1 hour digestion. (2) I take an aliquot and check the bands with control in agarose gel electrophoresis. (3) If there are still undigested, I will adjust and add more enzyme and extend the reaction time for a couple of hours more. (4) After I see almost 1 cut after hours of reaction, I will proceed to gel extraction. I believe I'm making sure or maximizing that there will be no supercoiled DNA.
Other option if you want to avoid the cut & paste old system is the NEB assembly kit, with this you design 2 couples of overlapping primers, amplify the vector and the insert in two PCR reactions and then you assemble them, no need for restriction sites and you can insert your DNA wherever you want.
- I will try to check this one out. Thank you!Following
- Kyle Cheung added an answer:1Does anyone have a gene construct for SirT3?
I am working on Sirt3 and mitochondria in neurodegeneration. Has anyone gene construct for Sirt3?
Hello Dr. Parihar,
In our lab we have a mouse model that overexpresses a SIRT3 construct as well as adenoviruses for both a full-length SIRT3 as well as a truncated SIRT3 which lacks a mitochondrial localization signal. Would this be of any interest to your group?
- Theo Nikiforov added an answer:9Any advice on why the restriction digestion is not working?
I have fluorescent labelled DNA oligos, having a single BamH1 site. I'm screening some compounds which may affect the enzyme cleavage efficiency. My problem is that the oligo is not getting cleaved at all even in the absence of my compound. There are 5 bases upstream to the cleavage site. I have varied oligo conc., enzyme quantity, and time. I would appreciate all leads in solving my problem. Thanks in advance..Following
- Jai Ghosh added an answer:1Could protozoan be developed as a novel expression system ?
Mammalian cell expression systems are utilized to produce special proteins that cannot be correctly folded or modified in Escherichia coli. However, the cultivation of mammalian cell is much more difficult than that of E. coli.
Protozoans, such as Paramecium caudatum and Amoeba, are microorganisms which are easy to cultivate. Bestides, these organisms are also belongs to animal. Therefore, is it possible to develop protozoans as an easy - to - cultivate expression system with the ability to efficiently and correctly express heterologous proteins ?
Try it and see....... You may even find sometime certain fungal cultures or yeast cultures too do the same job.Following
- Jahdiel Larraguibel added an answer:6Has anybody in India used GeneScript for the synthesis of their gene of interest?-
Biomatik ships internationally and has Indian distributors. Their gene prices are really great and the product quality is good. You can check out more on their custom gene synthesis page here; http://www.biomatik.com/services/custom-gene-service/gene-synthesis.html
- CHANDRA SHEKHAR DASARI added an answer:4Has anyone tried plasmid transfection with lipofectamine 3000 in a melanoma human cell line or another human cell line?
Hi, I need to transfect a melanoma cell line with a plasmid but I’m not sure how much DNA (ug) is necessary for the transfection and which plate is better for this, 96, 24 or 6-well plate.
I accept any advice, protocol and suggestions
We do regular transfections using L-3000. It works nicely. You can not experience cytotoxicity like others(L-2000) and simple protocol.
1)Take recommended amount of Serum free antibiotic free medium in tubes(equally)
2)Add P-3000(2"x"'ul) and "x"'ug of plasmid to one tube. For second tube add "x"ul of L-3000 reagent & incubate both for 5 min.
3) Mix both solutions and incubate for 10-20 min and finally add to the cell culture plate.
No need to change medium(10%FBS DMEM/RPMI with Antibiotics) before and after since it works based on CRISPR-cas9 method untill unless required. Plate type, duplicate or triplicate and DNA concentrations("x"'ug) depends on your experiment as Dr. Joel Mentioned. All the best.Following
- Julien Courchet added an answer:18What is a good vector mapping software?Can anyone suggest a suitable vector mapping software? Suppose I have a sequence of a vector, I want to know the promoter, terminator, antibiotic resistance etc. in a region. I used NEB cutter, but this one is useful only to track restriction sites and CDS.
Joining the discussion a bit late. I really like ApE as several users recommended above. Really basic but does the job. However I recently changed my laptop and it is now very slow and ineffective. Wonder if anyone has had the same issue?Following
- Jahdiel Larraguibel added an answer:11Which company for gene synthesis?I need a fungal gene synthesized, but I'm having trouble in choosing between the existing companies. I've been Google searching, and found two companies in the US that I took in consideration: Biomatik and Genscript. Problem is that I haven't found any customer reviews about them... I was wondering if any of you have some past experience with these companies, or if you could suggest a different company which is good (and maybe cheaper as well) for this purpose. Much appreciated!
I would second Biomatik for custom gene synthesis. Genes are for the most part fairly standard so what is important is turn around time and price, both of which are great at Biomatik. Even if you find a lower price elsewhere, they will price match it. Get more information here http://www.biomatik.com/services/custom-gene-service/gene-synthesis.html
- Jakub Červenka added an answer:3Could someone give me insight into why gene duplications could be occurring when targeting essential genes in S. pombe?
We are trying to make conditional mutants in essential genes of Schizosaccharomyces pombe, using the nmt1 promoter and different degrons, and antibiotic selection in haploid cells. The homologous recombination works and we get the expected insertions, but the lack of phenotypes has prompted us to check whether the genes were actually interrupted... and they are, but it turns out that a wt copy duplicates elsewhere in the genome (which explains why there is no phenotype).
In diploid cells, we don't obtain any positives after selection.
Could anyone give me insight about reasons for these duplications, and how to avoid this problem?
I agree with Petre Ianakiev. I had similar problems with essential gene in Saccharomyces cerevisiae and found out that this problem can be strain specific (different genetic backgrounds in different strains). I would suggest you to try different strain.Following
- Md, Asadur Rahman added an answer:18Why competent cells of E.coli DH5α cannot grow after transformation?
I have made competent cell of E.coli DH5α with CaCl2 method. This is the following method:
1. Grow the cell on 50 mL Luria Bertani Broth until OD600 between 0.3-0.6
2. Aliquote the cell culture until 1.5 mL into mini centrifuge tube
3. Incubate on ice 10 minutes
4. Centrifuge the culture at 13,000 rpm 4°C 1 minute
5. Discard the supernatant and then resuspension the pellet with 0.5 mL of cold CaCl2 0,1 M
6. Centrifuge the culture again at 13,000 rpm 4°C 1 minute
7. Discard the supernatant and then resuspension the pellet again with 100 μL of cold CaCl2 0,1 M
After the competent cell is ready, I want to transformation the cell with heat shock method:
1. Add 5 μL of ligation product into 100 μL competent cell
2. Incubate on ice 30 minutes
3. Move the tube to waterbath at 42°C for 45 seconds and then move into ice bath for 2 minutes
4. Add 900 μL Luria Bertani Broth into the tube
5. Incubate at 37°C 120 rpm for 1 hour
6. Grow into Luria Bertani Agar - Ampicilin 50 mg/mL medium
After used that method the result is negative (there's no one growth). Is there any mistake of that method?
I think the problem is with your ligation product. Try to transform some whole plasmid in to your competent cell and grow it with the appropriate medium. Then you can understand the efficiency of the competent cells. If the competent cells are ok, then give attention to your vector construction again. Cheers.Following
- Sabine Brandt added an answer:62Which part of the plant is most preferred for DNA isolation?
Different parts of the plants can be used for the isolation of genomic DNA. But which part is most preferred for DNA isolation and why?
- Pablo G. Goicoechea added an answer:2The possibility of matching one SSR marker with different chromosomes.
Can anyone tell me about the possibility of attaching an EST-SSR marker within 2 or 3 different chromosomes?
Apart from Alex answer, it is also possible (and rather common) to map a marker to more than one chromosome (linkage group) in diploid organisms. For recent genetic maps, made with lots of markers and mapping software, it comes down to the LOD scores. The cut-off of your LOD score is a compromise between type-I and type-II errors (i.e., to establish as linked a marker which is not, and not establishing as linked a marker which is actually linked). As in any statistical process, you can have some markers in the interface....You can solve it by getting a larger progeny or by, adding other markers which lie close to the region(s) where the problem marker is located.
Hope this helps
- Vinutha Kb asked a question:OpenIncrease protein half life by cloning at NcoI site in pET vectors?
How does cloning at NcoI site circumvent N end rule when the 2nd amino acid of insert happens to be lysine? would not the G after ATG in the Nco1 site CCATGG shift the reading frame of the insert?Following
- Melanie Vargas added an answer:11Triple transfection mechanism : does GFP testifies of efficient transfection when expressed ?
Pardon me if the question sounds a bit stupid but I just would like to be sure to understand the triple transfection mechanism. I'm performing triple transfection for AAV2 production on HEK293T cells using 3 plasmids : Rep/Cap, Helper and GFP. Do cells expressing green fluorescence testify that the triple transfection has been efficient, which means that the virus is TOTAL (genome + capsid) ? Or can I have fluorescence even if the virus is not total ? As the GFP is in the virus genome, can it be expressed without the virus to be encapsided ?
Thanks a lot for your help !
Thanks a lot this is very helpful !
I think I'm gonna consider indeed to check my ITRs on the GFP plasmid.
Thanks again !
- Chiara Benedetto added an answer:6Problems with RT-PCR (shRNAs testing)
I am testing some shRNAs by RT-PCR and I do not have hands-on experience with this technique.
In particular, I tested some primers and a PCR reaction for the very first time, but I obtained the expected signal just for the positive control (without shRNA). Before starting any experiment, I made sure that primers sequences were correct, as well as amplicon size, and I set an annealing temperature based on primers Tm.
I cannot justify my result by saying that all my shRNAs are working, because I do not have any signal in my shRNA control! What can I do to optimize my RT-PCR?
Many thanks for your answers.
actually the housekeeping gene yields the same signal in all the samples. What I did not manage to see was the signal in the "empty vector", but now I've optimized my RT-PCR and it works.
Thanks again for all your useful suggestions, they helped me a lot! And good research!Following
- Gregory Dressler added an answer:6How do I induce a specific mutation in a gene construct for creating a knock-in mouse model?
Hello, as a behest for the university we have to create a transgenic knock in mouse with a specific deletion of lysine 210 in the TNNT2 gene, which causes the development of dilatated cardiomyopathy. I was able to design a genetic construct which allows me to create a knock-in model for this gene. But now I have no clue about inducing the mutation in the specific exon. I read on the internet that a mutation can be induced by radiation, chemical agents and transposons. I hope one of you can help me with a way of inducing this deletion of lysine in the gene.
With kind regards,
I was just guessing. When you said dilated cardiomyopathy , I assumed that is a post-natal phenotype, i.e. it is not embryonic lethal which is important because the cas9 is so efficient that you will get many homozygote mutants even in the F0 generation.Following
- Bhoj R Singh added an answer:99+Concentration of glycerol used for storage of bacteria?I looked up many protocols for freezing bacteria, but the concentration of glycerol varies from 20% to 80%. How should I choose? Is it v/v or w/w? One of the protocols mentioned spinning the cell first and then adding fresh medium and 20% glycerol.
If liquid nitrogen is unavailable, is it ok to put the mixture from RT into a fridge at -80C? Sometimes cell pellets formed after freezing, how can I avoid pellet formation without using liquid nitrogen?
updated on April 9
I think I may fine some causes to the previous failure of my recombinant expression E.coli. Some colleagues suggest that there are different treatment for different types bacteria.
Sergii Pochekailov: as for a high-expression-rate strain, it is better to use freshly transformed bacteria, or the expression would drop dramatically.
Jen-Ning Tsai: in the case of the BL21 and its derivatives (commonly used in recombinant protein expression), the manufacturers suggest a lower glycerol concentration for storage of the cells, since glycerol concentrations (> 10%) may lead to plasmid instability.
updated on June 7
Ignasi Roca mentioned another interesting method for bacteria storage. Using skimmed milk instead of glycerol. Since scraping the vials without completely thawing the sample required a very fast operation, he change into 20% skimmed milk. He get longer survival times, besides, thawing is not an issue anymore! I f you are interested, the protocol is detailed in his answer and attatched paper.
In our lab we routinely use 25% glycerol broth to store culture at -20C, For more details on preservation of bacteria you may read page 31-33 at the following link:
- Robert Olinski added an answer:4How do you choose your Next Gen Sequencing (NGS) service provider?
I am starting NGS service business and would like to ask end users how and why they choose their NGS provider?
What would make them to switch to a new one? Price , Quality, Support , Turnaround time, Help with the experimental design ?
Thank you in advance, each opinion is very valuable!
Go for competitive price, quick delivery dates and proximity of client. If you target clinical NGS have Ion S5 platform in-house, if draft genome seqquencing take Illumina. Apart from DNA seq, expand to RNAseq, and 16SRNA seq, variant calling, SNPs detection, etc. Have a pdf report with all relvent statistics generated after every run for customer inspection.Following
- Biswanath Chowdhury added an answer:3Is it possible for any middle exons of a gene to start with ATG or end with TAG/TAA/TGA
Is it possible for any middle exons of a gene to start with ATG or end with TAG/TAA/TGA ? Shouldn't it behave as start or stop codon?
I think that TAG / TAA / TGA / ATG might be present at any position along with the start and end positions of a gene. But can it be present at start / stop of any middle exons?
Suppose a middle exon contains following nucleotide sequence.
Case 1 : ATGaacggattat
Case 2 : aataacggatTAG / aataacggatTGA / aataacggatTAA
Are the above cases possible? Please help and if possible provide me with examples/links of such exons/genes present.
yes it is possible that start and stop codon may present within exons but if we see that very carefully then we will see that stop codon is not present within the same frame of the transcript (joining off all coding part of a nucleotide). If we proceed by reading three nucleotides (codon) at a time then the stop codon must not present in that frame.
If you consider the above example then you can see the stop codon is not present in the same frame from where the coding region is started.Following
- Harald Lund added an answer:4Is it possible to inject tamoxifen into the brain of mouse of a Cre-Lox P?
I have a conditional gene knock out crossed with a tamoxifen inducible Cre system. It is a widespread cre line.
I wanted to specifically knock out the gene in the amygdala. I thought it might be possible by doing a stereotaxic injection of tamoxifen into the amygdala. Is there a precedent for this? I could not find any papers on this.
For my I.P tamoxifen inductions, I use tamoxifen dissolved in corn oil. But would I need a different preparation for the brain?
Curious whether you tried this successfully yet?Following
- Ricardo Furtado added an answer:23What is the best percentage of glycerol stock preparation for transformed culture recommended for the long term storage?What is the best percentage of glycerol stock preparation for transformed culture is recommended for the long term storage? How long will cultures be efficiency maintained?
I use a solution of 50mM CaCl2 and 15% glycerol, for storage at -80ºC. Now, I have a question myself. Would this solution affect my bacteria in a negative way, if they are kept overnight at 4ºC in it? I made this mistake myself, and my cells did not become competent, so I'm wondering if this would be the cause.Following
- Marcelo Augusto Szymanski De Toledo added an answer:6What is the efficiency of using Crispr to edit just a single allele of a gene?
I would like to use Crispr to edit just one allele of a gene, in mouse ES cells (by "edit" I mean that I'll provide external DNA fragment for HDR). I would like to keep the other allele of that gene, intact (meaning no indels or other mutations caused by NHEJ).
The external DNA fragment that I will be providing, will also contain Neo between the homology arms (for selection), and HSV-TK outside of the homology arms (for minimizing random integration by using ganciclovir).
At the end of the process, after adding ganciclovir and Neo, how many Neo-resistant colonies do you think I will have to sequence to find one in which one allele was correctly edited, and the other was left intact?
Do you think Crispr will add any advantage here versus not using Crispr? (If I won't use Crispr, and just transfect with the external DNA fragment, there is no risk of damaging the unedited allele... on the other hand, without using Crispr to provide the DSB, the chances of HDR will also be greatly diminished...)
1- indeed, i did not get any homozygous HDR clones, but one might consider that the mutation I am introducing might be lethal when homozygous
2 - we used the l-755507 to increase HDR as we considered that increasing the number of positive clones for HDR we would increase the chance of having heterozygous ones without indels in the other allele.
3 and 4 - yes, we used double nickase system in ES and iPS cells.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.