Rui:LabNotes/Collaborations/2011-5-20

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Additional 10 cycles of direct lysis samples[edit]

  • Continue with lab note on 5.19.11
  • Additional 10 cycles for direct lysis samples (sperm and ES) in parallel with 500pg RNA and 150pg gDNA

QPCR curve[edit]

File:Additional 10 cycles 5.20.11.jpg

TBE PAGE[edit]

File:10 cycles 5.20.11.jpg


Repeat 2nd strands and PCR with the remaining samples[edit]

  • Continue with samples on 5.19.11, the remaining 9ul A-tailing product
  • 2nd strand with P8
  • PCR with 30 cycles

QPCR[edit]

File:5.20.11 remaining samples.jpg

Beads purification[edit]

  • Combined into 4 samples, 8 PCR tubes per samples
  • Sperm RNA, ES RNA, Sperm lysis, ES lysis
  • 0.7X beads purification
  • Elute with 50ul water
Sample ID ng/ul 260/280 260/230
sperm RNA 43.64 1.9 1.68
ES RNA 295.67 1.93 2.18
ES lysis 220.08 1.92 1.97
sperm lysis 23.5 1.68 0.95

Cloning[edit]

  • Ligation: ~25-30ng purified PCR + 2.5ng vector in 10ul total ligation, 14C, o/n
  • Transformation, using Kan30 plate