Hello, I'd like to ask for your help because polycystronic vector cloning is not working well using 2A sequence.
The final product we want to make is as follows
restriction enzyme1 - Target1 - P2A seq - Target2 - restriction enzyme2
The first PCR with R.E1-Target1-P2Aseq and p2Aseq-Target2-R.E2 is going well, but,
Making a final product through overlapping PCR is not working well.
1. When I tried to make primer including P2A full seq, the primer length was too long and the Tm value was also too high. So I randomly cut the 2A seq part and put it in each primer (The 2A sequence overlapping between the two primers is about 20bp), is it possible that the overlapping area setting is wrong? If so, how should I fix the primer?
2. As I told you before, the Tm value was so high that I had no choice but to make a primer by dividing some of the sequences. Is it okay to make a primer by ignoring the high tm value and putting the whole 2A sequence in?
3. Why is it that bands are sporadically made in multiple sizes when overlapping PCR?
4. We are testing various annealing temperatures such as 60, 65, and 68, but please advise if there are additional variability conditions that can be applied to the experiment.
5. If any researchers have experience with overlap PCR, I would appreciate any tips or precautions about the experiment!
No one around me has ever done polycistronic vector cloning, so I'm asking you this question. A little advice would be very helpful. Thank you!
Hi, having a larger overlap-more than 20bp might help.
Also how are you setting up the overlap PCR?
Hi Tae-Yong Jeon
I agree with Purva in that the overlap area must be at least 20bp.
Also, choose the highest possible annealing temperature from your temp. gradient experiment to get rid of the sporadic bands.
Coming to the PCR set up, for overlap extension, it is generally done in a way that first 10 cycles the templates are allowed to denature and renature at overlap areas, and after that stitching step is over, Polymerase and primers (end primers) are added. The next extension phase can go for 20-25 cycles.
Hope it helps,
Subham.
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