- Mohammad Nazim added an answer:How many site directed mutagenesis base changes do you have to change to make a construct resistance to a siRNA?
I am trying to do a rescue experiment so need to mutated my construct so that it is not knocked down by my siRNA. I've got the sequence and just need to design primers for the site directed mutagenesis. But one base pair change didnt seem to be enough to make the construct resistant to the siRNA.
Can anyone suggest how many changes to make
2/3 or every amino acid in the sequence?
Recently, I was successful to make a siRNA resistant cDNA construct for my experiments. I have introduced 4 silent mutations, at the 3rd base of four consecutive codons. If it doesn't work, you can increase the number of silent mutations.
2/3 mutations also might work, but with lesser possibility of success.Following
- Jordan Robin Yaron added an answer:Does anyone have some experience/useful advice for isolating single clones?I am currently trying to isolate single clones, post-transfection with a GFP expressing plasmid so that I can evaluate the efficiency of the CRISPR technique. I tried single clone isolation by dilution and FACS in a 96 well plate, but I don't get any fluorescent cell that survives long enough to do the clonal expansion (I know the plasmid fluorescence is transient but normally, within the first few days, I should see it so that I can let a colony grow from it). I suppose the FACS may stress them too but I think it would be more reliable than the dilution...
Any suggestions/ideas are welcome!
Kris, can you explain more about your imaging setup? What sort of microscope? What sort of culture vessels? Transfection method? Cell type?Following
- Hideyo Yasuda added an answer:What prevents FLAG from expression in the plasmid?
I am having a struggle with the flag tagged expression vectors. I have constructed a flag-tagged TR using the PCMV6 myc-DDK vector from Origene. The flag tag was fused in frame at the C-terminal of the ORF of TR (stop codon removed). However, when I transfected it into the 3T3L1 cells, no expression of flag was detected. I thought it might be a problem with the vector. So I moved the ORF of TR into the sigma p3Xflag-CMV-10 vector, this vector has the 3Xflag at the N-terminal. However, still I can't detect any flag by western. And I have a flag-tagged protein as positive control. The sequencing showed no mistakes. Has anybody encountered a similar problem before?
Thanks for update of your experiment. You finally have got good results by your hard work.
I think both promoter and stability of the protein with tag affect the level of the protein in 3T3L1 cells. The combination of CMV promoter and flag-tagged protein is not enough to accumulate the protein to be detected by flag antibody in 3T3L1 cells. The EGFP-tag at N-terminus might stabilize the protein so much but flag-tag might not.
Anyway I hope you could get excellent results by use of EGFP-tagged protein.
- Thomas E Darga added an answer:How can I determine why my siRNA is differentially knocking down my two mRNA isoforms?
I ordered siRNA duplexes from origene, and the sequence of all three recognize a region which is common between the two isoforms of my mRNA. In theory, this means it should knockdown BOTH isoforms as a result. Instead I see that siA only knocks out isoform B, while siB and siC knock out isoform A.
The two isoforms only differ in exon 3, and all three siRNA duplexes target regions in exons 6 and 8.
These experiments were performed in HeLa cells which express much higher levels of isoform B than A.
I was wondering if anyone knew what could account for these differences in isoform preference for knockdown, when the siRNA should target BOTH mRNA sequences.
Another possibility is that your cell line has SNPs that interfere with siRNA efficacy and/or detection. Cell line SNP and other information is available from the Broad Institute's Cancer Cell Line Encyclopedia at the link below. HeLa cells do not appear to be included in CCLE, but the pubmed link may help.Following
- Negar atashi shirazi added an answer:Which 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?
- Swaranjit Cameotra added an answer: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.
I think 10% glycerol solution should work well for you.Following
- Yuan-Yeu Yau added an answer:How are antibiotic resistance genes useful as markers for genetic engineering?
I know the resistance genes are attached to a gene you want, and then the cells are treated with an antibiotic, and the surviving cells have taken up the genes. But I thought antibiotics would not harm plant cells, so why would the plant cells which have NOT taken up the gene act any differently than those which have?
We use those antibiotics or herbicide which WILL harm the native plant cells for selecting transformants (only the transformed cells will survive selection). Some of the antibiotics will not harm the plants (under a special condition), such as the antibiotics we use to suppress/ eliminate Agrobacteria after plant genetic transformation.Following
- Muhammad Naveed added an answer:What is the effectiveness of glycerol to preserve bacterial cultures at -80C ?
I want to ask, for how many time (years), a bacterial culture remain preserved with 40% glycerol at -80C ?. In the other sense if a culture remain in glycerol for 2 years then it have any effect on the efficiency and activeness of strain or need to refresh culture after every 6 months?.
Yup, it is all right, you can preserved it up to one year in this conditions.
Just maintain cold conditions like -20C or for better at -80C and glycerol 20% to 40% is ideal for this regards.
- Darius Balciunas added an answer:Any advice on the sequence of the msi1 gene promoter in zebrafish?
I took 2500bp upstream the first exon from ensemble and i checked in ucsc browser but it doesn't have CpG island, so how can I get the sequence for this promoter? can I trust that this 2500 contains the promoter sequence? can anyone help me please?
Knowing how complex gene regulation is, 2.5 kb is rather unlikely to be complete but it may be sufficient.
One can invest a lot of time in computational analyses of a putative promoter. Or one can just PCR it up, clone into a vector with a fluorescent reporter and actually see if / where it drives expression. That's what matters in the end anyway, right?Following
- Sham ashok Patil added an answer:What 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?
what are the alternatives to -800c?Following
- Hanno Loubser added an answer:How can I confirm Agrobacterium (GV3101) transformation?I am facing problems in confirmation of transformed GV3103. Transformed bacteria is able to grow on antibiotic selections media (Rif+Gent+Kan) while the wild type was unable to grow. But colony PCR and PCR on mini preps were negative. What can be the problem? I tried different concentrations but still the bacteria was able to grow.
Your colonies might just be yeast. We have a big problem with this due to the fact that Agrobacterium cultures have to grow for long times. How does the Agro colonies look? Colony PCR is difficult with Agrobacterium. Spin down 1 mL of your overnight culture and resuspend it in ddH20. Then boil it for 5 min and use 5uL of that in your PCR reaction. Works every time :-)Following
- Nisrine Nisrine added an answer:Vector containing eukaryotic elongation factor 1 alpha (eEF1alpha) promoterI have to clone and express a viral gene in a mammalian cell line so I'm searching for a shuttle vector including eukaryotic elongation factor 1 alpha (eEF1alpha) promoter with multiple cloning sites plus neomycin & ampicillin resistance gene for selection. Does anyone have experience with such a vector?
Can you tell me please wich primers we can use to sequence EF-1 alpha promoter vector ?Following
- Yudistira Wahyu Kurnia added an answer:Does 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.
Hi Sarah, I think the one from addgene (which is pmaxFP-Green-N) mentioned by Ganapati is different with pMAXGFP (yours). And I think those restriction sites written on the map are the only unique restriction sites can be used for single digestion.
Actually I am now facing the same problem with you.
I don't know whether you still work for this now anyway, but I have suggestions for you even if it would take longer time and less chance of success.
You can digest in one of the restriction sites in pMAXGFP plasmid that you want to use and do blunt-ing followed by dephosphorylation. Next, blunt end your GOI (without dephosphorylating it) and insert it into your linear blunted-dephosphorylated pMAXGFP plasmid.
Finally after cloning, you have to screen the correct direction of your GOI by double-digesting the restriction sites inside your GOI and one from pMAFGFP (I will explain further about this if you want) or just read the sequence using your GOI sequence primers. Kind of not so good idea but I would perform what I mention above as well soon. I appreciate if you have better suggestions for me.Following
- Rhiannan Hope Williams added an answer:Is 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?
I tired this to get specific expression in the cortex, easy hit right, and it didnt work. I didnt use corn oil but a different graded oil which was nice and clean (and works for i.p). We injected very slowly and the recovery was very delayed. It seems tamoxifen is likely a prodrug so its needs to go i.p. to work.Following
- Lech Kaczmarczyk added an answer:Should CRISPR/Cas9 donor sequences be linear or supercoiled?
I plan to introduce a specific alteration into cultured cells using CRISPR/Cas9. I am cloning 1kb homology arms (2kb total) into my vector. Should I use the intact supercoiled vector for transfection along with my guide + Cas9 plasmid? Or a linearized vector, or the isolated linearized fragment containing only the homology arms and the targeted alteration? Thank you very much.
We always linearize HR template vectors while doing gene targeting. Are you going to select for stable clones following your transfection? In any case, I would suggest to linearize your vector, ideally with 2 restriction enzymes (could be next to each other) to prevent re-ligation. In my opinion, with a circular vector you have a higher chance for O-type recombination, which is not what you want to achieve.Following
- Suraj Kumar Mandal added an answer:Does cloning vectors (pET series specially) confers toxicity to the gene/protein of interest?
I am working on a thermophilic protein which is not known to be toxic. It was earlier cloned in pET11a and cell death was observed after 2-3 hrs of IPTG (0.1mM) induction. I then cloned the gene into pET22b and it worked for me. No cell death was observed and protein was well expressed. What could be the probable reason for toxicity in pEt11a?
Thanx everyone for your valuable suggestions. I have got my protein soluble in BL21 DE3 pLysS. Cloning the Gene in pET22b proves to be a correct decision as @thansanqa @Arhtur and @gaurav had mentioned the reasons.Following
- Jahan Dadgar added an answer:What are some efficient methods to clone multiple guide RNAs into a CRISPR/Cas9 nickase expression vector?
What are some efficient methods to clone multiple guide RNAs into a CRISPR/Cas9 expression vector like pX462? How can they be optimally implemented in practice?
I was considering the "Golden Gate" technique but I have no experience with this. Anyone have some advice for the multiplexed CRISPR wannabes out there?
Here is a link to the plasmid: http://www.addgene.org/48141/
It has ampicillin, puromycin resistance and BsbI cleavage sites.
i have no experience with paired nickase approach but would like to try it. Why not co-transfect the GuideA-pX462 and the GuideB-pX461 and the repair template all at the same time and screen the GFP isolated clones. Very interested to hear your answer.
Have you had any success creating a vector with multiple guide sites?
- Nishant Neel added an answer:What 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.
You can do this on Benchling. Just drop your sequence file in or paste in the bases. Customize what appears on the map (e.g. annotations, primers, cut sites) and export it as a PDF or SVG when you are done, like this example: https://benchling.com/s/bo6DLuaGFollowing
- Mathieu Dondelinger added an answer:Can someone help regarding plasmid dna sequence?
I have a sequence written 5'>3' from the people that made a plasmid with an inserted dna that codes for a protein. how do I know if the sequence they gave me is going forward or backward? I am trying to figure out if changing a certain base in the sequence would change the amino acid it codes for not. Going one way it does, one way doesnt. How do I know which direction the promotor is going in?
For example if top was the sequence they gave me. How would I figure out if the amino acids the DNA codes for is AAT then TGG or if it is CCA then ATT.
Traduct your gene of interest : sequence is on the same strain of the start.Following
- Huong Nguyen Lan added an answer:May anyone help with gene knockout in Actinomycetes bacteria ?
I need to construct Micromonospora mutants using gene knockout. I have never done this before. I just do some search online, and find a method so far:using the insertion of a suicide vector (a plasmid harboring a selection marker, which is not able to replicate in the target strain). I was wondering if anyone know which method is better or if there are other good methods?
Thanks in advance and waiting for your reply
- Floris Schoeters added an answer:Is it possible for an E. coli DH5α with pEX18Gm to grow in a medium containing ampicillin?
I found that E. coli DH5α with pEX18Gm established colonies in medium containing ampicillin during an experiment. According to Hoang et al. (1998), pEX18Gm contains only three selectable marker genes, carbenicillin, gentamycin, and tetracycline. Can anybody help to explain this phenomena?
- Richard Christison added an answer:Please help I am new in Plasmid construction, can any one tell me, is my design right or wrong?
First , if i understand that i will buy new plasmid for mammalian transfection.
Second, Go to NCBI website take the cds (orf) for my gene on mRNA
Third check two restriction enzyme will not cut my gene and cut only in MCS
third,Design the primer and I really Confused in this step ,Now I need to take care in designing because i Need to put three things GFP , Two restriction site
so, in my forward primer I will put one ( first restriction site followed by bases complementary with my gene ) , reverse primer i will put 2nd restriction site + bases complementary with my gene)
So, Where should i Put My GFP sequence ?
Some people told me take care about 3 frame when you design What does this mean
There are many ways you could go about construction of a fusion, and as Ferdinand has highlighted, whether to use a N- or C- terminal tag is protein dependent. If your protein is a membrane associated or secreted protein a C-terminal tag will be required so as not to disrupt any signal sequences.
You may be able to find an expression vector that suits your requirements that already has a GFP cloned into it, in which case you just need to design your PCR primers in frame and clone. If you can't then I suggest you create vectors with GFP in them, then clone your gene into them. You might find it easier to design 2 vectors, one for the N-term and one for the C-term. Note an N-term construct should have your cloning site(s) in front of any stop codons.
Another thing to think about when designing your constructs, is to ensure there is a good Kozak sequence - (G/A)nnATG(G) - around the ATG start. The -3 G or A is vital to good translation from the start codon, the +4 G is less so but if you can have it, it is good to include it.
- Malachi A Blundon added an answer:Has anyone seen increase transcript production from fly mutants generated from p-element mobilization?
I have a mutant fly line that was generated by inserting a p-element in the 5' UTR of my gene. My RT-PCR suggests the transcript is elevated in my mutant. I am confident in my controls; they look correct. I'm just wondering if anyone has observed this before?
Thank you everyone for your responses. To answer your questions, Sudipta, gal4 is not in the background and this is an EY element. The pcr is performed on embryos from homozygous mutant mothers mated to homozygous mutant fathers. It turns out the increase in transcript is not statistically significant after running three biological replicates. We have decided to take an alternative approach to answer our question. Thanks again!Following
- Dmitry Guschin added an answer:Can anyone provide insight regarding Scr7 for enhancing genome editing?
Two publications from March in Nature Biotechnology show that Scr7 can be used to favour homology-directed repair (HDR) over non-homologous end-joining (NHEJ) for CRISPR/Cas9 applications.
I've been trying to repeat this in K562 and HEK293T cells (one of the studies used HEK293 cells), but so far haven't seen any effects. I tried 0.01-10mM Scr7, adding the drug immediately after, or 6h/12h after electroporation and then replenishing the drug after 24h and 48h. But none of the conditions I've tried so far have had any effect compared to my control.
I was wondering if anyone might have experienced troubles with Scr7 stimulation as well?
Could a standard media compound (DMEM,10%FBS,10mM HEPES, 1% Pen/Strep) affect the drug activity? (I'm about to try antibiotic free medium next, but my hopes aren't high on that one)
The publications used Nucleofection or Fugene for their plasmid transfection. I am working with classic electroporation as we don't have access to a nucleofector and found that Fugene is not suitable for the delivery of single stranded HDR templates.
My electroporation buffer is high in potassium salts, and is diluted 1:20 in culture media after electroporation. Could salts from the buffer or cell debris from the electroporation step interfere with my drug's activity?
I would be very grateful for your input!
Not being en expert on this, but it seems is not easily reproduced. See some notes for example here: https://groups.google.com/forum/#!forum/crispr . If something can affect the drug cinc. in the media, the FBS will be my first suspect. May be trying a FSB minus condition after EP is worth checking.Following
- Besra Samy added an answer:In DNA extraction from saliva, why doesn't the pallet dissolve in the tube after adding DDH2O?
I am extracting DNA from Saliva through Manual Protocol NOT with the Standardized Kits. Right at the end of the extraction when I get the DNA Pallet that need to be dissolved by adding de-ionized water which normally does dissolves and we process it to further step gel electrophoresis. But I am facing difficulty in this step that is the pallet which does not dissolved. I am using Phenol, Chloroform, EDTA, Tris, BME and PK and Lysis buffer. Some of my colleagues ask me to wash the Phenol with TAE Buffer solution this will help you in proper extraction. Kindly help me out in this regard.
Subin Cheri Kunnumal Raj I used to do that, but recently after overnight incubation @ 4C, the pellet get swollen & not that easily to be dissolved by pipetting . Do you suggest anything to overcome swollen pellet?Following
- Nicolas Grandchamp added an answer:Do you have any suggestions on virus toxicity after lentiviral transduction?
I am using lentiviral transduction system. I produce virus in HEK by calcium phosphate method. However, I don't have fluorescent marker. The only marker in my plasmid is neomycin. When I transduce the cells, they start to die 24 hours?, however, there is no problem with untransduced controls. Thus I cannot proceed to neomycin selection. I don't think it might be related to polybrene concantration because I always use the same concantration with this cell line before. So what do you think about the problem? What can be done to solve it?
Agree with the old posts, it could be due to the nature of your transgene.
Another parameter to check is the optimale concentration of G418 to use because it is not always the same for all the cell lines.
When we do Neo transductions with a new a cell line, or even with a new batch of G418, we realize a range of different concentrations and select the lowest concentration where all the cells die (generally between 0.8-1.7mg/ml).
Hope this help you,
- Kumarasamy Jothivel added an answer:Problem in induction of clone gene in a pET 32a vectorI am trying to clone 2 Kbp amylase gene into pET32a vector, after sucessful cloning into vector and transformation into BL21(DE3), it was checked by colony PCR and Restriction Digestion analysis, which confirm presence of my insert. The reading frame is also correct since I used a software to design primer which takes under consideration of vector and insert also, all these are ok. My problem starts when I try to induce, after induction using IPTG at 0.1-5mM conc at 0.6 OD for 6hrs I am getting in both the induced and non induced protein band pattern similar - on checking the activity of the transformed host cells and the lysate supernatant on a starch agar plate, it shows a clear zone of hydrolysis but I am not able to detect on SDS PAGE. Please suggest if any one could help me out with this problem.
try auto induction medium for inducing the cloneFollowing
- Michael J. Riviello added an answer:Any suggestions on the reporter fusion construct using CRISPR or Tol2 in Danio Rerio?
I have very little experience in this. I would like to develop a reporter assay, by fusing GFP to the response element of a particular nuclear receptor. I wanted to accomplish this using either CRISPR or Tol2, and was wondering if it is necessary to generate a fusion construct consisting in GFP and a proximal promoter, or if it is possible to to insert only a GFP sequence upstream of the regulatory element using the CRISPR-Cas system? I apologize if it seems silly.
The papers are extremely helpful, thank you!Following
- Srinath Rao added an answer:Can anyone has a gene for inducing dwarfism in banana, how to get it?The banana cv. I work with grows 14ft. in height
Can anyone has a gene for inducing dwarfism in banana, how to get it?
Thanks a lot. Nice papers very useful to meFollowing
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.