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PCR: Big Primers = Big Problems (for me) - I'm really not sure what's going on in those poor little tubes... (Jun/21/2008 )

Hello, I've been having a bit of an issue with a pair of primers for PCR.

One is a 60-mer and the other is a 49-mer. I'm planning on using the primers to do a "sticky-feet" mutagenesis to insert a protein's sequence before another protein's sequence in the plasmid I'm using. The reason for the difference in size is I'm also inserting a 4-amino acid sequence to cleave the first protein from the second (I need a control where the two are separate for my experiment).

The problem I've been having is on the first of two PCR steps I'm just trying to amplify the sequence I want to insert by PCR which means that my two primers are using a 26bp sequence on both of their 3' ends, the other bp's being unbound and needed for the actual muagenesis PCR.

I've tried a couple of things all with no success: (note: I'm using PrimeSTAR DNA Polymerase by Takara which recommends much shorter annealing times 5-15 seconds by default)

98C: 10 seconds
55C: 5 seconds
72C: 14 seconds
(30 cycles)

This resulted in no products at all.

Next I tried samples in parallel to normal with 5% DMSO under the following conditions:

98C: 10 seconds
50, 55, and 60C (in parallel, used a temp gradient): 15 seconds
72C: 14 seconds
(30 cycles)

This resulted in some lanes showing a plasmid-length PCR product (my desired product is ~225bp long) as well as a faint band at ~100bp

Finally, I tried touchdown PCR dropping from 60C to 50C at 1C decrements and an additional 25 cycles under the above conditions, this led to a similar lack of bands other than plasmid length...

The Tm's of my annealing regions on my primers are 60C and 64C (long and short respectively), if that helps...

I'm quite inexperienced with PCR and I'm hoping someone can help me as I'm at wit's end...

Thanks in advance!


Just suggestions:

1. do more gradients, in fact a gamut ranging from 50 to 66. Ignore your tms; once you run into trouble, all bets are off.

2. When doing this, increase teh number of cycles to 35. Once you find the right temp, you can get back to less cycles.

3. Increase the extension time to 30 sec. Not going to do any harm, but may make it work.

4. I hope 98'C denaturation you are using is according to manufacturer. If not, do it at 94.

5. If your system allows it, design two new primer pairs. There is no sense in wasting too much time on PCR optimization when primers are so cheaper and quicker to get.

6. Since you claim to be inexperienced with PCR, you might be doing some common mistake/s, so do a regular easy PCR working the lab, so you know you are adding everything right etc.



Thanks, I'm gonna try out some of those now; I'll report back biggrin.gif