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]
TBE PAGE[edit]
File:50ng Nextera PCR 5.23.11.jpg
Library construct by ligation with Y adaptor[edit]
Procedure[edit]
- Digestion with BciVI in 30ul or 50ul (SL)
- Beads purification 1X
- Ligation with Y adaptor (1:10):1.1ul, 0.7ul, 0.5ul, 0.7ul
- Beads purification (up to 50ul, 0.7X)
- 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]
PCR failed, highly doubt if Y-adaptor is too low (invisible in the gel?)