Blue:RNA-Seq Experiments:07022014: Difference between revisions
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==NexteraXT== | ==NexteraXT== | ||
''Nextera Tagmentation'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|Tagment DNA Buffer||||||||||2.5ul | |||
|- | |||
|Amplification Tagment Mix||||||||||1.25ul | |||
|- | |||
|Diluted Sample||||||||||1.25ul | |||
|- | |||
|Total||||||||||5ul | |||
|- | |||
|} | |||
::*Vortex and spin | |||
::*Incubate 55C 10 min | |||
::*Hold 10C | |||
::*Once sample reaches 10C add 1.25ul of NT buffer in block | |||
::*Vortex and spin | |||
''Nextera Amplification'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|Tagmented Sample||||||||||6.25ul | |||
|- | |||
|Nextera PCR Mix||||||||||3.75ul | |||
|- | |||
|S501 Primer||||||||||1.25ul | |||
|- | |||
|N7id(1-24) Primer||||||||||1.25ul | |||
|- | |||
|Total||||||||||10ul | |||
|- | |||
|} | |||
::''Mix and centrifuge'' | |||
::{| {{table}} | |||
|- | |||
|72C 3 min|||||||||| | |||
|- | |||
|95C 30s|||||||||| | |||
|- | |||
|95C 10s -> 55C 30s -> 72C 60s||||||||||12x | |||
|- | |||
|72C, 5min | |||
|- | |||
|} |
Revision as of 16:58, 10 July 2014
PBMC C1 Experiment
Extracted RNA from bulk samples using Zymo microRNA Kit and eluted in 6ul. Determined [RNA] using Qubit:
- CD4+ 26.1 ng/uL
- CD19+ 6.27 ng/uL
- PBMC 95.8 ng/uL
Diluted to 1ng/ul and used 1ng as tube control for C1 run 20140702 (followed standard Smart+randomer C1 tube control protocol with 3 fewer cycles in last stage of PCR)
Qubit quantification of PCR product:
- CD4+ 6.56ng/ul
- CD19+ 7.60ng/ul
- PBMC 5.02ng/ul
- Neg Cont 1.64ng/ul
NexteraXT
Nextera Tagmentation
Volume Tagment DNA Buffer 2.5ul Amplification Tagment Mix 1.25ul Diluted Sample 1.25ul Total 5ul
- Vortex and spin
- Incubate 55C 10 min
- Hold 10C
- Once sample reaches 10C add 1.25ul of NT buffer in block
- Vortex and spin
Nextera Amplification
Volume Tagmented Sample 6.25ul Nextera PCR Mix 3.75ul S501 Primer 1.25ul N7id(1-24) Primer 1.25ul Total 10ul
- Mix and centrifuge
72C 3 min 95C 30s 95C 10s -> 55C 30s -> 72C 60s 12x 72C, 5min