Athurva Gore/LabNotes/2011-2-18: Difference between revisions
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* Loaded as: Low-Mass Ladder, 100 pg, 10 ng, NTC | * Loaded as: Low-Mass Ladder, 100 pg, 10 ng, NTC | ||
[[File:02182011_Low_Input_Klenow.jpg|600px]] | [[File:02182011_Low_Input_Klenow.jpg|600px]] | ||
* Both libraries seemed to work well | |||
* '''Klenow appears able to replace the Nextera extension step''' | |||
==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 |
Revision as of 19:20, 18 February 2011
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
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