PCR and templates - (Jan/28/2010 )
I have a question regarding PCR and the nature of the template:
1) Good amplification if I use as template a previous amplified insert.
2) No product if the template is a miniprep product containing the insert cloned in a vector.
Why sometimes, not always, I am not able to get any PCR product using the second option.
Thanks in advance
Could it be your template concentration in option 2 too low? have you check the template concentration before running the PCR?
Could it be your template contain inhibitors carried over while you do your miniprep? Why don't you check the concentration and the purity of of template? And try to do some dilution of the template if the concentration is sufficient. If the template concentration is low, why don't you try to increase the number of cycle of your PCR reaction?
Thank you for replying. I agree with you, my thought is that may be my minprep product concentration is too low. But I guess that the concentration of a miniprep product (even with low-copy plasmid) should be much higher than the PCR product used as template in option 1. Anyway, i will quantify both. Thank you for your suggestion.
To test Option 2, try a 1/10 dilution of the template in MQW.