PCR Cloning-large primers - (Jul/08/2009 )

Hello,
I'm trying to clone a gene using a plasmid as a template. I have one pair of primers designed to insert a Not1 restriction site and a HA tag to the gene. Also I have another pair of primers designed to make a truncated protein with an Eco restriction site and a HA tag inserted in the middle of the sequence.
My questions are: which Tm of the primers do I have to consider for the reaction?? As in the first cycle of reaction only a part of the primer (the one that is complementary to the sequence) the Tm range from 62-66ºC. But from the 2nd cycle the whole primer will be complementary so the Tm is higher (~82ºC, yes, Celcius, not Fahrenheit, I know.....).
Do you have any suggestion on how to achieve this cloning? I have tried to amplify it by PCR at annealing temperature from 50-68ºC without success. I have also tried with different Mg concentration, hot-start, different denaturation time (up to 30s). I am desesperateeeeeeeeee!!! please, help!
Thank you

Are you giving the elongation enough time? TAQ polymerases at a speed of ~60bp/sec, do the calculation for the amount of time you need and then give an extra 5-10 sec per cycle.

I'd do at least 5 cycles at the low Tm before I'd go up in the temperature.

The Tm is the temperature at which 50% of the primer is annealed.

I don't see a reason to go to the whole primer Tm (82) because if you work on a clean plasmid you don't have to worry about unspecific priming of the uncomplimentary part of the primer (no need for the high stringency).

Another thing you may want to consider is making your primers long enough. When you introduce restriction sites and tags, you are introducing a lot of sequence that has no complementarity. If the instability introduced by the non-complementary part of the primer is higher than the stability provided by the complementary part of the primer, you will not get any amplification no matter at what temperature you anneal.

Many times you can save yourself time by designing multiple primers and testing them together rather than performing a lot of troubleshooting on a single primer set. Primer design can be very tricky, and the rules about how much non-complementary primer you can have and still get PCR amplification are sketchy at best.

You can try adding betaine (1M) and /or DMSO (I use up to 10%).

Have you tried touchdown PCR, where you start with a high anneal, and drop it by ~1 degree per cycle for 10 cycles or so?
How different are the Tms of your primers? You might want to extend the lower temp primer to within 4 degrees of the other one. Also, how did you determine the Tm?

Sally- on Jul 8 2009, 09:34 AM said:

Any time I've done this sort of amplification, I run the whole PCR using the annealing temp of the gene-specific sequence, not including the restriction site. MOST of the time I'm able to get a pretty clean target band, but once in awhile there is some non-specific amplification. I don't think it would hurt to raise the annealing temp after a few cycles, but I've never found it necessary, particularly if amplifying from a plasmid.

Occasionally, I've had primers that just don't amplify well. I'm not always sure of the cause, but regions with high A/T or G/C composition may cause you fits. It doesn't even have to be the region where your primer is, just the DNA around the annealing site can cause problems.

Double check the primer sequences to make sure they're correct and use an oligo analyzer (I like the one from IDT: http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/) and be sure to check for self-complementary sequences. If you're adding restriction sites, by virtue of the palindromic sequence, you're already going to have 6 complementary bases in your primers for self-annealing. Also check for hairpins in your primers.