I've been trying to amplify 3' splice variants of a hormone receptor gene, and I initially had great success. However, now I cannot repeat my results. The first 3-4 PCR's ran great, but now I only get a smear. I have replaced my primers because I was worried they were contaminated, but I still get the same smear. When I use different primers as a positive control, I get good clean amplification, so I think my cDNA is still OK. I need this specific reaction to work so I can amplify enough DNA to sequence the splice variants - any thoughts?
Hi,
Here is a possible explanation of your results:
During the first PCR reactions everything worked fine but because of freezing/thawing cycles or some DNase contamination or... your cDNA started to degrade by the ends. Then your control primers (I assume they are internal fragments) still work while your race reactions don't because they need the RACE primers target sequences that are in the ends.
To test that I would design specific primers near the 3'end based on the sequences you got in your initial reactions and try again.
you can also try gradient PCR just in case for some reason the reaction conditions are not the same than in the initial reactions (change in any reagent, mistake in annealing temperature of your initial reactions or...)
good luck!
Hi Nick!
I'm missing a little bit of data here: How do you perform your RACE experiment? Also, if all you need is cloning the variants, why did you need to return the experiment so many times (you say the 3-4 reactions went just fine), i.e., what was the difference between the 3 first reactions and the following ones?
Best,
Yehu
Depending on which step you're up to with your RACE, you can try lowering the amount of DNA you're putting into your PCR reactions. I find that the less DNA I have in my reactions, the smaller the smear and the more specific my product are.
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