To all the brilliant scientists and lab techs out there,
I am preparing DNA library for NGS illumina, using SPRI beads purification method. My input gDNA is 500 ng. I've recently noticed that my DNA yield after PCR purification is low.
Here is the protocol for my PCR clean up using SPRI beads.
1) 1.1x SPRI beads is used for the clean up. post PCR product + SPRI beads incubation time is 5 mins.
2) Magnetic rack separation time: 2~3 mins. (until the solution turns clear).
3) Remove supernatant
4) carefully wash the beads with 70% ethanol twice. ( The SPRI beads is not disturbed during washing)
5) dry the beads for 2 mins. ( not too dry as you lose DNA)
6) elute with water. (2 mins incubation)
I'm just curious what could be the reason my DNA concentration is low after PCR clean up? btw, the beads was brought to room temperature before any use.
As per request, my expected length is around 400 bp.
Thank you so much for all yall help
In order to provide any kind of answer you will need to provide the expected length of the your PCR amplicons.
If your expected size of the library post PCR is less than 200 bp its bound to happen as ratio your using for clean up is more suited for longer amplicons. Try using a higher ratio such as 1.5x or 1.8x and you will see definite improvement in the yields. As Jonas has mentioned in the earlier answer if you provide expected length then it would be easier to answer your question.
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