Rui:LabNotes/Collaborations/2011-5-23

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Library construct by Nextera[edit]

Procedure[edit]

  • 50ng samples: sperm RNA, ES RNA, sperm lysate, ES lysates
  • 5ul tagmentation: 0.25ul transposon, 1ul LMW buffer
  • 50ul Kapa FAST PCR: barcode adapter 2 (ID9-12), 10 cycles
  • Beads purification, elute in 20ul

QPCR curve[edit]

File:Nextera 5.23.11.jpg

TBE PAGE[edit]

File:50ng Nextera PCR 5.23.11.jpg

Library construct by ligation with Y adaptor[edit]

Procedure[edit]

  1. Digestion with BciVI in 30ul or 50ul (SL)
  2. Beads purification 1X
  3. Ligation with Y adaptor (1:10):1.1ul, 0.7ul, 0.5ul, 0.7ul
  4. Beads purification (up to 50ul, 0.7X)
  5. Phusion PCR with N2.R.ID5 - ID8

Concentration[edit]

>Digestion ng/ul 260/280 260/230
sperm RNA 39.12 1.9 2.77
ES RNA 51.57 1.92 1.92
sperm lysis 16.27 1.73 1.42
ES lysis 45.64 1.93 1.99
>Ligation ng/ul 260/280 260/230
sperm RNA 35.03 2.05 2.19
ES RNA 45.66 1.92 1.99
sperm lysis 13.99 2.04 1.7
ES lysis 39.23 2.06 2.12
>PCR ng/ul 260/280 260/230
sperm RNA ID5 21.29 1.73 1.69
ES RNA ID6 21.31 1.72 1.7
sperm lysis ID7 19.24 1.61 1.14
ES lysis ID8 20.03 1.74 1.1

QPCR curve[edit]

Original 10 cycles: File:Phusion 10 cycles 5.23.11.jpg

Additional 5 cycles: File:Phusion 5 cycles 5.23.11.jpg

TBE PAGE[edit]

File:5.23.11 Phusion PCR.jpg


PCR failed, highly doubt if Y-adaptor is too low (invisible in the gel?)

Cloning of PCR product[edit]