Primer concentraion

[Research assistant]

Hi all! I'm currently working on cloning the gene for CFTR, a chloride transporter, but I can't seem to get any band on a gel after running a PCR, and I am fairly certain that there is something amiss with my PCR.

I have tried running a dilution series of my cDNA, but with no luck. I am currently running a gradient PCR which should tell me something about the optimal annealing temperature, but I got a tip that increasing the primer concentration and shortening the annealing time night provide a better result. However, Ive never attempted this before, so I'm hoping someone has done it before and can give me a tip or two!

My primers are currently used in a 10 microM concentration, which I assume is pretty low.

Have any of you tried this before? If so, what concentration of primers did you use? And did you shorten the annealing time? What about annealing tempreature?

[kmunson779]

Typical PCR primer concentrations should be from 0.1 to 1 micromolar (according to several manufacturer's websites as referenced from Google). 10 micromolar seems way high to me.

I'd try a lower primer concentration to see if that makes a difference.

[danfive]

This is how I set up my PCR primers:
Stock Primers 500X = 500microM
Working Primers 50X = 50microM
Primer Concentration in PCR reaction is 1 microM

For regular PCR, optimization usually leads me to adopt final primer concentration in PCR reaction betwee 0.4-1microM.
In case or real-time PCR 0.1-0.3microM (final conc) is very common.

If you have a recent PCR machine, (doesn't require mineral oil over PCR reactions), then the annealing temperature can be shortened considerably (for example 20-30sec). Two step PCR (skipping the annealing temp) is not the next logical step in PCR optimization, unless you have the traditional PCR already working very well (with a lot of PCR product).

If you have a very low expressed gene, then it could likewise take many PCR cycles to produce visible PCR product. Try playing with the DNA concentration in PCR reaction, consider nested (or semi-nested) PCR. Also consider BSA(final conc 1mg/ml) in the PCR reaction as an adjuvant.