primer Tm is too high, how tu get pcr product - help! (Oct/14/2010 )
Step 1: try your pcr with an annealing temperature of 55C.
ElHo on Fri Oct 15 10:08:38 2010 said:
What kind of template are you using in your pcr? We usually design primers with a lenght of 20 bps for genomic DNA. Adding a restriction site plus some additional bases at the 5`-end (for efficient cutting of the pcr product), you will end up at approximately 30 bps. But as laurequillo already stated, only the matching part of your primer are used for Tm calculation. You could also try to do more than 30 cycles in your pcr. But this also augments the danger of misincorporations.
thank you！I will have a try！
laurequillo has a good point, you should only use the 28mer that is complementary to your template to calculate the Tm. I think you will find this drops the value considerably.
I would do as phage434 and run your PCR with a 55C annealing and see what happens. You could even run your gradient starting at this temp or there abouts.
phage434 on Fri Oct 15 11:53:46 2010 said:
Step 1: try your pcr with an annealing temperature of 55C.
Thank you,I have already pcr at this temperature but it didn't work.
jane-00 on Sun Oct 17 08:37:20 2010 said:
phage434 on Fri Oct 15 11:53:46 2010 said:
Step 1: try your pcr with an annealing temperature of 55C.
Thank you,I have already pcr at this temperature but it didn't work.
What you mean by it didn't work? at 55C you see no bands, but you mentioned previously at 60+ there are faint bands?
You can try a long range gradient 55C to 65C.
Also, what is the calculated annealing temperature for each of your primers? Only the matching site without the RE added sequences?
Or, try to excise the faint band and do a pcr on it?
Another idea would be to make a pcr buffer with lower salt concentrations. This effectively lowers the Tm of your primers.
I also agree that a "nested" pcr may help your yield. You cut out the faint band from your first pcr, purify and use this as template in a second pcr reaction. I've had this greatly increase yield in difficult reactions.
In my experience sometimes changing the polymerase can work wonders. I for example have PCRs which don't work with Phusion PCR, but work wonderfully with Dynazyme-Polymerase and vice versa.
Besides varyiing the temperature, you could also try to vary the amount of DMSO you use (from 0-10%) or as previously mentioned the buffer system. E.g. for Phusion-Polymerase there are a HighFidelity- and a GC-buffer (for GC-rich sequences) available.
If you get faint bands that's a good start. Try using this temperature while changing other parameters of the PCR reaction. With a little bit of luck one of this conditions will work just fine. If nothing helps you probably have to design ne primes (preferably with as little secondry structure as possible and no dimer formation).
adrian kohsf on Sun Oct 17 16:14:48 2010 said:
jane-00 on Sun Oct 17 08:37:20 2010 said:
phage434 on Fri Oct 15 11:53:46 2010 said:
Step 1: try your pcr with an annealing temperature of 55C.
Thank you,I have already pcr at this temperature but it didn't work.
What you mean by it didn't work? at 55C you see no bands, but you mentioned previously at 60+ there are faint bands?
You can try a long range gradient 55C to 65C.
Also, what is the calculated annealing temperature for each of your primers? Only the matching site without the RE added sequences?
Or, try to excise the faint band and do a pcr on it?
I finally get the product at 60℃ by LA taq with GC buffer, but I have to clone it to the express vector,the fidelity of LA taq is not enough,so have you some advice for me,thank you!
Try redoing the pcr with a high fidelity polymerase but using the GC buffer. Keep all pcr conditions the same as when you saw product.
How do you know the fidelity is not enough, when you can get your band at 60C?
According to the manufacturer:
http://catalog.takara-bio.co.jp/en/product/basic_info.asp?unitid=U100005352
It says: "Under general PCR conditions, higher efficiency and higher fidelity are obtained compared than conventional Taq DNA polymerases. This enzyme is basically applicable to any template DNA, and especially effective in amplifying more than 15 kbp fragments."
Also, you mentioned your product is 770bp. Based on the takara manual for GC buffer: http://catalog.takara-bio.co.jp/en/product/manual_info.asp?unitid=U100005352
only when you clone fragments >5k it will have lower cloning efficiency. Thus, I don't feel there is any problem for you.
Under worst case scenario, you might just do a few more tubes of PCR, gel excise all the bands and pool it into a single tube. This "might be" sufficient enough for you to do your cloning work, at least I had done something similar before for clone and express my fragment.