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Old 07-13-2016, 08:47 AM   #4
melop
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Location: college station

Join Date: Jan 2012
Posts: 22
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Today I performed more tests. I adjusted the fragmentation step to get a bigger gDNA distribution. I also left the beads in after ligation clean-up for PCR. This seems to help with the size distribution a bit. The longest fragment size I can get now is 372bp (insert 222 bp). However the sheared DNA should have a peak at 354bp, plus 150bp of adapter that should be around 500bp. So my final library is still 130bp shorter than expected...

One thing I notice is that when I ran the ligated fragments, the Y-shaped adapter makes them run slower. But there seems to a second, lower peak. Are these unligated fragments? If so perhaps I can increase adapter input (currently I'm using 1ul of 5uM adapter mix in 20ul reaction) to make sure they are ligated?
Alternatively, perhaps the End-repair and A-tailing wasn't efficient enough, so the longer fragments in the library don't get the adapter ligation?

Any pointers would be appreciated!
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File Type: png sheared_beadselected.png (78.9 KB, 13 views)
File Type: png library_ligation.png (139.2 KB, 10 views)
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