I want to introduce a single mutation by overlap PCR using a pair of 20-mer primers (complements of one another) containing the mutation plus two 5' and 3' end primers. In the first PCR (extension PCR), I will get two fragments of 1500 bp each. In the second (overlap PCR) I will mix these two amplicons followed by 15 PCR cycles. Finally, I will add the end primers to the mixture and continue with 15 additional cycles. I wonder if fragments (1500 bp) are too long for the overlap PCR to work.
1.5kb is definitely doable. Myself have done recombination of two 2.5kb fragments before. However, there are a few considerations:
1. the size of the overlap. When I did my 2x2.5kb recombination, I used overlap about 50bps. For yours 20mer might be a little too short, but you never know until tested.
2. the PCR process. You don't have to separate your PCR into two sections. In my experiences, sometime you can do it with only one end-primer (5' or 3') for your recombination PCR (or overlap PCR). Just think that one of your 1.5kb fragment is a mega-primer. I usually just do a test run with one 5' or one 3', or both primers in three separate reactions, then pick the one with the best results.
3. PCR conditions. To run PCR in a single un-interupted reaction, I usually run first 3 cycles at 55C, then raise the annealing temperature to the whatever standard temperature your primer(s) require. The first three low temperature cycles are critical for synthesizing full length (~3kb) fragments which will serve as the main templates for later PCR cycles.
Good luck!
1.5kb is definitely doable. Myself have done recombination of two 2.5kb fragments before. However, there are a few considerations:
1. the size of the overlap. When I did my 2x2.5kb recombination, I used overlap about 50bps. For yours 20mer might be a little too short, but you never know until tested.
2. the PCR process. You don't have to separate your PCR into two sections. In my experiences, sometime you can do it with only one end-primer (5' or 3') for your recombination PCR (or overlap PCR). Just think that one of your 1.5kb fragment is a mega-primer. I usually just do a test run with one 5' or one 3', or both primers in three separate reactions, then pick the one with the best results.
3. PCR conditions. To run PCR in a single un-interupted reaction, I usually run first 3 cycles at 55C, then raise the annealing temperature to the whatever standard temperature your primer(s) require. The first three low temperature cycles are critical for synthesizing full length (~3kb) fragments which will serve as the main templates for later PCR cycles.
Good luck!
Thank you so much for your posts. Kejun!!!, I got a beautiful 3-kb amplicon following your suggestions. Hope the sequence is OK!!!
The key is the extent of overlap. Typically we use at least a 30-mer with the mutation in the middle. Finally 3 Kb is not too long
I have used quikchange mutagenesis kit previously, and it works literally the first time around, and you can have your mutant construct in a day!
I tried it the old school way of using overlapping primers, but i had to troubleshoot a lot, and was taking too much time.
If this is something you are going to be doing often, i would highly recommend using the quikchange kit
In that case follow the Quikchange protocol; not clear why you have 2 PCR products?
Do people have a sense of how much of each PCR fragment is being added to the overlap reaction? mass or molarity?
Kapa Hifi DNA polymerase is better than Phusion. Instead of performing overlapping PCR, seamless cloning kit of Invitrogen is a good alternative. It needs only 15 nucleotide similar in each ends.
In fact, I used Kapa HiFi and worked well with 20 nucleotide ends.
1500 bp is no problem at all. I did a overlap PCR with 3 fragments about the same size in one reaction and it worked perfectly. Good luck.
MG
I did and doing quickchange in lab. I think that is best 1 step method. Anyway, goodluck
Hi Gontzal,
I have recently done an overlap PCR joining two DNA fragments of 600 bp and 3000 bp respectively and it worked well. As you will know already you need to use a proofreading DNA polymerase (Phusion (Dynazyme) for example) to generate blunt ends in your two fragments and likely gel-purify them (it works better).
The only difficult thing may be to reamplify your PCR product if you need more for downstream applicactions. Not sure why but reamplification of a PCR product (even after gel extraction) is sometimes a problem for long fragments (other people reported the same problem described also in the literature). In this case you could carefully "extract" your PCR product from the gel with a tip and then rinse it into 30 microliter of dH2O. Then you use it as template to avoid carrying with you non-visible non-specific PCR product which will likely be a problem for the reamplification.
Max.
Add both PCR product in equimolar ratio and perform OE-PCR in single step followed by a nested PCR. Single step PCR: Denaturation-2min, Annealing-Tm - 5 C for 3 min and Extension - 72 C for 15 min. Use this step PCR amplicon/s as template in next PCR using Forward Primer of Gene 1 and Reverse primer of Gene 2.
It ll work fine....
I've developed a website to assist in this sort of primer design, so you might find it useful: www.rf-cloning.org
You can also check out my NAR web server pub relating to the site @ dx.doi.org/10.1093/nar/gks396
Take care.
I don't feel any problem to amplify pcr product of 3kb length by your strategies. But I want to suggest one thing that, if this mutated product if your using for the protein expression purpose then do use the taq polymerase with proof reading and as high fidelity to avoid further consequences related pcr generated errors in pcr amplification using normal taq polymerase..........All the best.................
Theoretically yes. It should be better to use regular Taq polymerase for overlap PCR. However, you also increase the probability of mistakes in other regions (not intent for mutation). For all my overlap (recombination) PCRs I have done (believe me, there were many), I used Phusion Taq (from Thermo/NEB) for all and I had successes for all of them. And the reason is that the mutations are already incorporated into your fragments in the first round of extension PCR. By the time you do your overlapping PCR, the overlapping regions are identical, there is no "mutation" involved.
Plus, I prefer the Tm+3 for annealing temperature and short extension time of Phusion Taq. With a fast ramping PCR machine, it can greatly shorten the time required for P CR reactions.
Kejun Guo, i am runing an overlapping PCR with Phusion from Thermo Neb, may you send me the program you use..I use primer AB and CD to generate two pcr products corresponding around 250 bp of the gene upstream and downstream. My B and C primers contain complementary bases around 25. Then i use the pcr products of AB and CD as template in the second round, i run PCR with Pusion also for 7 cycles then i add the primers A and D and run for additional 30 cycle. What i get is a weak band of the overlapping product which i can´t clone. my annelin tempreture is 40 for 30 sec before the primers and 55 after i add the primers.
Thanks
I have used Q5 for overlap extension but I am not very happy with the result. It's fine if there are only 2 fragments, but when I try to overlap 4 fragments I only get a huge smear.
I used Q5 also and I don't have any problems to join three fragments (each of ~1 kb ). I think PCR conditions and the length of overlap is important. Q5 works the same as Phusion does but Q5 is more convenient to use because it use 2x reaction mixture that have everything there.
Hi,
20 bp is just fine. I routinely do OL-PCRs with just 15 bp overlap and sew 3 fragments in one go. Run 15 no-primer cycles at 55 oC annealing (40s) and 72 oC (1 min), followed by 20 Purification cycles with primer. You'll definitely get it. Through this method, I have obtained products as large as 4 kb too.
Good luck :)
Hi
does anyone has protocol for overlapping PCR using phusion polyemerase HF ?
thanks!
- Badges
- Science method