Hi guys, I'm here to ask for help!
We met a very frustrating problem!
We tried to combined 4 fragments (include vector) by Gibson.To this end, we added 30 bp overlap at the two sides of each fragment by PCR using Q5 hot-start HF polymerase.
We designed super-long primers for this purpose (60 bp).Then two of our fragments (their templates were purified PCR product) could get very bright bands. But we can never get any PCR product from the other two (one of them is EGFP sequence from another plasmid, the other one is just linear pGEM T easy vector).
We tried three times by using different annealing temperature from 55C to 65 C (the annealing part's Tm is ~60C, and the whole primer's Tm is ~65C).
I'm wondering what would be the problem here? Are there anyone could give us any suggestion or commands?
ps: It would not be the template's problem, at least for the plasmid one. We have used the same template but different primers (no add-on sequence, only ~22 bp long), no problem at all.
Thank you!
Yours,
Jennifer
Hi Jennifer,
are you sure that there is no mistake in one of the primers sequence? As primers for generating the overhangs for Gibson assembly always are very long, it is recommended to have them HPLC purified. But be aware that with this method only the correct length but not nucleotide sequence is checked. I already experienced several times that just reordering a primer (giving the same sequence) cures all the problems. Of course, check also with your vector map.
Hope this comment helps! Good luck!
Gertrud
Hi Gertrud,
Thank you for your suggestion! So you mean, even with the same sequence, sometimes we may just get the incorrect primers? Yes, we have to order some new primers.
Thank you!
Yours,
Jennifer
Indeed, at least with our supplier it sometimes happens that the nucleotide sequence of the primer is not correct.
Gertrud is right, there can absolutely be quality problems with primers, particularly with primers that long.
I may be commenting out of turn without seeing your design, but if your primers are for Gibson assembly (or any other ligase-free DNA joining), you don't need them to be that long. Primers with 15-20 bases for PCR annealing and 15-20 for "overlapping" the adjacent fragment give you 30-40 bp overlap between adjacent PCR fragments - way more than you need for either ligase-free DNA joining or overlapping PCR. Ordering shorter primers will save you money on HPLC purification (you won't need it) and can reduce primer-dimers and hairpins that may inhibit PCR efficiency.
If your Gibson assembly is not very efficient with shorter overlaps, use a better system, e.g. Seamless cloning (Life Tech / GeneArt) or In-Fusion (ClonTech) - they recommend only 15 nt overlap
First of all I would also suggest reordering the primers. Maybe the company had made mistakes, maybe your water for reconstitution was not DNase-free, maybe...
Another suggestion for not-working PCRs is not only to play with temperatures but also with Mg-concentration or other additives like DMSO, running many cycles with low annealing temperatures. Once you get bands you can start optimizing further.
I can't imagine with a 30 bp overlap the Tm difference would be just 5C. Either your overlap is shorter or your Tm calculation is false, or you're in a crazy A-T rich region that you should avoid as a matter of principle.
So, first, get your Ta calculated correctly, for the "annealing" part and entire primer. Then, step up the Ta: Start with 3 cycles using the "annealing" part Ta. Then, run the rest with the higher, whole-primer Ta. (in the later cycles most each cycle's template will be a product that anneals to the whole primer).
As a followup, we have solved the difficulty, as described in
Preprint Rapid, modular, and cost-effective generation of donor DNA c...
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