I have been trying to amplify DNA isolated from PBMCs. However, after PCR on running samples on AGE, I am getting smears on my gel. I have been using 1% gel and TAE buffer.
Having not seen the gel i will assume that the cycling conditions are not stringent enough and you are obtaining non-specific amplification. You can improve the strigency by either increasing the annealing temp or by adding divalent magnesium to your reaction. It could also be that your primers are not specific or were incorrectly stored, resulting in oligo fragments. Lastly, i have seen this occur when the gel is reused or buffer is contaminated. I recommend making fresh buffer using ddH20. If it is still occurring then, try raising the annealing temp. If it still occurs, order new primers.
Hello Vanshika
First and foremost check the DNA concentration that you are using because smears can form with high concentration of DNA. Then try keeping your cycle number within 25-35.
Some changes in the PCR conditions will also have to be made like raising the annealing temperature or reducing the extension time.
There are other aspects which you will need to look in to like reducing the primer concentration, optimizing the Mg2+ as well as the nucleotide concentrations.
Thank you.
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