Athurva Gore/LabNotes/2011-2-18
Jump to navigation
Jump to search
Low-Input Transposase-Based Library Preparation
Klenow Extension Test
- Ran gel from yesterday's experiment
- Loaded as: Low-Mass Ladder, 100 pg, 10 ng, NTC
File:02182011 Low Input Klenow.jpg
- Both libraries seemed to work well
- Klenow appears able to replace the Nextera extension step
- Maybe wait a bit longer in next protocol? Waited 20 minutes, but have the appearance of lower efficiency.
- We could probably increase this by waiting longer
Bead Capture Experimental Plan
- Repeat Klenow protocol from yesterday
- Instead of going to PCR step, add TdT and TdT buffer with Biotin-labeled dATPs
- Capture DNA with beads, wash, amplify
- Check supernatant for DNA after Nextera PCR; run gel on supernatant
TdT Extension Test
- TdT is not very efficient with double-stranded blunt-end DNA
- Options:
- Denature before labeling; KOH - HCl denaturing?
- Increase incubation time suggested by protocols
- Found one dsDNA labeling protocol, but states that it is inefficient
Robert Lab Meeting Presentation Notes
- Look at base pair enrichment in: Lee W, Mutation spectrum revealed by paired genome sequences from a lung cancer patient, Nature, 2010
- Compare to what we found with Sergio's study