I am trying to clone full length integrin alphaIIb and have ordered primers from a publication. The forward primer binds about 20 bp upstream from the ATG and the reverse primer binds just after the stop codon, the gene is 3200 bp long. I have tried numerous different polymerases, increasing and decreasing the annealing temps, added various amounts of DMSO and tried Taq polymerase from Qiagen with the addition of Solution Q that I find amplifies any difficult product. So far I haven't even got a smear or hint of a PCR product. Obviously my extension time is long enough for a 3000 bp product and in theory that is not too long for PCR. I have other internal detection primers for sequencing and mRNA detection as follows: For 748, For 2200, Rev 2400 and Rev 2900. ALL combinations of forward and reverse primer (including the one that binds after the stop codon) work EXCLUDING the forward one that binds at -20 bp from start codon. I have ordered 2 further forward primers that bind 13 bp upstream of the ATG and one that bands at the ATG but still no product.
I have tried heat denaturing for longer before the PCR in case GC rich repeats, etc at the 5' end but that also didn't work. The gene number is NM000419.3. The first 300 bp is about 65%GC, so quite high but not that it should give absolutely no product - any other suggestions welcome.
I have also tried 3 different cell lines that were positive for the internal fragments in case of mutations in the promoter region.
Dear Mark
Have you also tried different MgCl2 conditions? It helps sometimes to lower or increase the concentration. I usually try PCR between 1.0 mM and 2.4 mM MgCl2. In you cas I would give a try with 5% DMSO, because the start region is high in GC.
Have fun
Rolf
Hi Mark,
if nothing of the above helps and the cloning of this gene is very important, you could also consider to let a company synthetize the fragment of interest. We recently did this in our lab with a similar problem and this speeded everything up a lot. This is still quite expensive but can save a lot of time, if this is a critical point.
Greeting
Kai
Hi Mark.
Just to get this right, have you tried these forward-primers also in other combinations (with internal reverse primers) and they worked in none of the combinations?
Ulrike Pfreundt, that is correct, none of the 3 forward primers that bind around the ATG work with any of the 3 reverse primers. I am not getting a truncated product or even a smear.
Rolf Marschalek, I have tried various DMSO concentrations up to 10% (so including 5%) and still nothing. I have tried various MgCl2 concentrations up to 2 mM, could try 2.4 but am not hopeful.
Hi Mark,
You should check if the sequence is correct. Are you relying on the sequence from the paper? I guess you double checked the sequence with online data? Ask the authors of the paper how they used their primers.
Regards,
Chris
Hi Christopher, thanks for the reply. I did a blast search and all 3 primers (published + own 2) around the ATG show 100% homology with the human sequence so not an error in the publication. I have also sent an email to the authors of the paper requesting info and not heard back from them. At a loss with this one.
To me this indicates very much that it's probably NOT the PCR conditions that don't work. It must have to do with the template.
What template are you using? Purified DNA?
hello
we amplify a 4,5 kb and 6,6 kb with touchdown PCR (very very long PCR), and after heminested or nested PCR. when put the 2 first PCR products on a gel, we can see nothing, or lots of bands. the second products (of near 1,5 kb) are great.
as Krzysztof Wicher said, sequencing is ok with 4 fragments.
regards
Ulrike, the template is cDNA that I made from cell lines. Controls such as GAPDH and b-actin work fine and the dact that I get all other fragments of my GOI suggest the quality is fine - I am also assuming template but have no idea how to solve the problem.
Melanie, I need to clone the full length gene to transfect cells - not just sequence as that is published, however I sequence all my cloned genes to avoid hassles at a latter date due to sequence errors. As the gene is 3200 bp I ordered the other primers so as to get overlapping sequences. I initially had 2 primers as a quick screen for +/- cell lines. When the PCR for the full-length gene didn't work I used them in the PCR to work out where the problem was - as it turns out the 5' end more than likely of the template.
Dear Mark
When I checked the Integrin alpha IIb = CD41 gene, I realized that there are 3 major transcripts. The longest one is 3333 nucleotides, while the other 2 are shorter (3232 and 1212 nucleotides). Both longer variants display the same exon/intron structure and differ only in a skipped 3'-exon. The first 2 exons are really high in GC, which may explain your experimental results.
I suggest to you another - and quite simple - solution: go to this website
http://www.lifesciences.sourcebioscience.com/genomecube?kw=CD41
and buy an "IMAGE full sequenced cDNA clone for CD41" for only 121 Euro.
I used this service some time ago when I faced a similar problem in the lab. The cDNA fragment comes within an mammalian expression vector, but can be subloned in any other vector of your choice. Saves time and money....
best regards
Rolf
what do you use to generate your cDNA ? we use Long range kit ? for a good quality of a whole 11 kb cDNA.
In my opinion it's much safer to use DNA and do a fusion PCR first amplifying the exons you want and then fuse those PCR products in a second step. If you need help with primer design dont hesitate to contact me. With cDNA you dont know whether you actually reverse-transcribed the whole mRNA. And whats more, RNA is a tricky thing. Assume a strong secondary structure in the region of the ATG (not that rare), then cDNA-synthesis would most probably not proceed over it.
Best, Ulrike
Thanks everyone for your comments, looks like Genome Cube is the quickest and easiest way to go.
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