Athurva Gore/LabNotes/2011-2-18: Difference between revisions

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(Created page with '=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')
 
>Ajgore
 
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* Ran gel from yesterday's experiment
* Ran gel from yesterday's experiment
* 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]]
* 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

Latest revision as of 19:21, 18 February 2011

Low-Input Transposase-Based Library Preparation[edit]

Klenow Extension Test[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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