Rui:LabNotes/SingleCell/2011-5-23: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>RuiLiu
>RuiLiu
Line 1: Line 1:
==Library construct by Nextera==
==Library construct by Nextera==
===Procedure===
* 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===
[[File:Nextera_5.23.11.jpg]]
===TBE PAGE===
[[File:50ng Nextera PCR_5.23.11.jpg]]


==Library construct by ligation with Y adaptor==
==Library construct by ligation with Y adaptor==

Revision as of 23:39, 24 May 2011

Library construct by Nextera

Procedure

  • 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

File:Nextera 5.23.11.jpg

TBE PAGE

File:50ng Nextera PCR 5.23.11.jpg

Library construct by ligation with Y adaptor

>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

Cloning of PCR product