PCR cloning strategy - (Jan/25/2007 )

Hi,

I am trying to clone a gene into a mammalian expression vector (pcDNA3.1). I need to add an N-term 6X his tag, a kozak sequence, and an Nhe I site to the 5' end and a BamHI site to the 3' end. I have decided to this by 2 rounds of PCR. However, I have had problems with getting enough product yield. I have tried different annealing temperatures(57-60), and have played around with template concentration(50-200 ng), primer concentration, number of cycles (30-40) etc. but nothing seems to help. Below are the primers I am using. Any suggestions?

5' CAT CAT CAT ATG GCG TCT TCC GTG GG 3' Tm= 61.2 C forward primer ------> This adds 3 Histidines on the N-terminus

5' TAG GGG ATC CTC AGG CTG ACT CAG TGA CTT 3' Tm= 64.6 C reverse primer ------> This adds a BamHI site on the 3' end of the gene.

I have used 2 different polymerases (FideliTaq from USB and PfuUltra from Stratagene). I keep getting very low yield. When I run 1/10 th of the reaction on a gel the band is extremely faint (but I can see it under longer exposures).

In the past I have gotten much better yields (1/10 th of the reaction shows up very brightly on the gel usually).

Any suggestions?

Thanks,
Roshni

Is there a spelling mistake NheI... there is no NheI site in the foward primer. NdeI site, yes. No NheI site.
Also look at the histag. I don't think you have 6 his. The primer only has sequence for 3 His codons.

Concerning these primers, which is the actual template binding sequence. The annealing temperature of your primers consider only the actual sequence that binds to the template. Not the entire sequence of the primer. I would recommend you recalculate the annealing temperature

I have no idea what the binding sequence actually is, but as the His codon are definately not part of your sequence, I would guess that you should start looking at an annealing temperature around 52 Celsius and below
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Because I am adding a lot of sequences on the 5' end, I am doing the PCR in two rounds otherwise my primer would be too long and the overhang will be longer than the part that actually anneals to the template. Therefore, I am only adding 3 his residues the first time around. In the next step I will add 3 more his residues + a kozak sequence + an Nhe I site.

I will recalculate the Tm for the part that actually anneals and try that. Thanks!

actually there is no need for that. I have done many PCRs with 100bp long primers, where the template binding segment is only 25bp. That works fine.

A few bits of advice here:

You don't need to worry about how much sequence you are adding to the 5` end of your primers or how long your primers are. You could easily perform this PCR in one round. I also add restriction sites and a Kozak sequence to every gene I amplify and I've had primers well over 50 bases that work perfectly fine, so there really is no limitation there.

The main thing to ensure is that the annealing temperature at the 3` end of your primers is high, so that you can use a high annealing temperature to ensure the specificity of your PCR. I commonly make my primers around Tm = 72C to make sure the annealing is specific.

Also, like Pernese says, the annealing temperature of your primers is measured using the portion of the primers at the 3` end that anneals to the template, not the whole primer. That is important because in the first cycle that is the only portion of the primers that will anneal to the template. If the Tm of that portion of the primers is too low they will not anneal to your target at the annealing temperature you are using in the first few cycles of your PCR and the first few cycles of your PCR will not work. In your case ATG GCG TCT TCC GTG GG and GG CTG ACT CAG TGA CTT are the sequences you should be using to calculate the Tm because these are the sequences that are binding to your template. That is only 17 bases on the 3` end of each primer. Don't be scared to use 25 - 30 bases if necessary to bump the Tm up to 72C.

Most importantly, I'm not sure how you've calculated the Tm for your primers but they are way off and I think this is why your PCR is not working well. Using Finnzymes Tm calculator http://www.finnzymes.fi/tm_determination.html, which uses the nearest neighbour method (the best method for calculating Tm), the Tm for your forward primer is 67.7C which is fine, but the Tm for your reverse primer is 53.7C, which is way too low for the annealing temperature of 57-60C you are using. I believe this primer is your problem - your reverse primer cannot anneal at the annealing temperature you are using.

You've got 3 options I can think of:

Use a lower annealing temperature and see how well the target is amplified.

Re-design the reverse primer so that it has a higher annealing temperature.

Better yet, redesign both primers so that you have sufficient Tm on the 3` end of each primer and so that all the sequence you require is found on the 5` end of each primer and only one round of PCR is required.

Good luck, Rob