Rui:LabNotes/Collaborations/2011-5-20: Difference between revisions

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==Additional 10 cycles of direct lysis samples==
==Additional 10 cycles of direct lysis samples==
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* 2nd strand with P8
* 2nd strand with P8
* PCR with 30 cycles
* PCR with 30 cycles
===QPCR===
[[File:5.20.11_remaining samples.jpg]]
===Beads purification===
* 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
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample ID'''
| align="center" style="background:#f0f0f0;"|'''ng/ul'''
| align="center" style="background:#f0f0f0;"|'''260/280'''
| align="center" style="background:#f0f0f0;"|'''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===
* Ligation: ~25-30ng purified PCR + 2.5ng vector in 10ul total ligation, 14C, o/n
* Transformation, using Kan30 plate

Latest revision as of 18:21, 17 June 2011

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