The results of quality control for one of my amplified libraries showed two peaks. The one at 200 was what I expected but I don't know what is the second one and if that might cause problem with sequencing?
The second peak may represent single-stranded library products that have self-annealed. This may occur because too much starting material was used at the beginning of library preparation or because too much template was used in the PCR reaction step. if the second peak represents a significant percentage of the library, it may affect accurate quantitation of the sample. In this case, you may want to quantitate the sample with a method that detects only double-stranded DNA (pico green, qPCR). You may also re-purify the library by running a gel and cutting out the band of interest.
Hi Katherine,
That was interesting point. I am certain that i didn't use too much DNA for library prep. But that is a good idea to check for the double stranded DNA. Thank you.
If the starting material was used more, then reduce a bit in the PCR cycles.Gel purification is good if the number of samples are less.
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