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Old 11-15-2010, 03:24 AM   #1
Junior Member
Location: Beijing

Join Date: Nov 2009
Posts: 3
Default Need help: Weird size selection

I didn't do size selection after adapter ligation and extracted DNA by QIAGEN PCR purification column because my material is very limited.
After PCR I didn't find band at 120bp as suggested by protocols as self-ligated adapter dimers.
my chipped DNA fragment is around 150bp before preparation, so after pcr 90bp should be added, then the DNA should be around 240bp?
I pcr 18 cycles and run 2% agarose gel and then cut at 200-300bp(see the attached image). I ligated these products to a vector and did transmission to E.Coli and sequenced some colonies.I found the DNA inserted inside the flanking adapter sequence were mostly less than 100bp.Why? Is it because the gel resolution is not good and small fragments show larger molecular weight? Should I cut longer fragment from the gel?
Thanks for your patience and hope you can give some advice.
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