Sunday 10/20/13

Posted on October 19, 2013 by admin

1:00p-2:30p, 5:00p-11:50p

Goals:

Today

  • write up some notes on Stowers presentations.
  • layout slides for Rowland Center talk (in progress)

This Week

  • Write up Banff Report!

Probe Making

PCR:

  • Large Regions (primer concentration)
    1. D11 100 kb percicentric Green (320 ng/uL)
    2. D12 385 kb Yellow (360 ng/uL)
    3. E01 200 kb Green (420)
    4. E08 75kb Red (420)
    5. E10 98 kb pure yellow (440)
    6. E12 630 kb Green (450)
    7. G09 76 kb Blue near ANTC (550)
    8. Negative control (62)
  • PCR performed with 1:60 dilution of original library
  • Poor PCR amplification. amplified another 4 rounds. Rerun gel.
    largeNoKpcr

PCR cleanup

  • D11, D12, E8, E12. low yield (20-30 ug/uL. E8 which looks the weakest has the most at 48 ug/uL).
    This one looks better.
    largeNoKpcr_try2

Try again, PCR

  • Spec primer concentrations down to 10 uM (for 40 bp T7 primers = 132 ng/uL)
  • Spec primary concentration down to 10 uM (for 20 bp = 75 ng/uL)
  • use library 9/22 1:20 dilution, dilute further 1:4 (so 1:80 final)
  • 50 uL scale PCR

Deep seq library

  • PCR to add illumina primers
  • pool 2 run with both primers 11 and 12.
  • request expense code to be able to sign up for qPCR machines
  • gel extract PCR
  • test gel a little messy but should do for gel extraction. Not sure what happened to sample #1 (A1).

Ph project

  • copy individual panels to powerpoint for Ajaz
  • export data from matlab to excel for Ajaz
  • select new cell
  • Unclustered Pc was suppressed in image, added back clear effect visible in mutants now.
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