pcr amplification after screening

NGS annotated.png

image: 2 ug gDNA loading per lane, annealing gradient between 61 - 65 C

Hello all,

I am amplifying my library for NGS sequencing after screening and seeing results as shown above. My concerns are that there is a large unexpected band <100 bp, likely primer dimer or something of that nature, and that my band may be a bit smaller than expected. It is worth mentioning that I have previously titrated my gDNA and found 2 ug to be optimal, which is why I have only used 2 ug in this reaction.

Has anyone resolved similar issues, or do you think I can move forward with amplification and pooling at 61 or 63 annealing temperature?


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Lime Golem11 months ago

try to increase the number of cycles

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