Rui:LabNotes/SingleCell/2012-12-17
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Hs Ref RNA with totoRNAseq ranging from 1ng to 10pg
- Repeat procedure used on 11.8.12/11.10.12
- set 2 condition prior to RT: 72C 3min vs 10C 10min
- set 2 condition for TSO: TSO.r02 vs TSO.idXX
Samples and procedure
- PNK: 1:2d in water
- PAP:1:4d in water
- RNaseIn: 1:2d in water
- MMLV:1:2d in water
' | RNA | Frag. | T20.id | ' | Anneal | after RT | TSO | N2.id |
X2 | 1ng | 1min | 1uM | id01-02 | 72C, 3min | 1uM | ||
X2 | NTC | 1min | 1uM | id07-08 | 72C, 3min | 3.2ng | TSO.r02 | N2.id01 |
X12 | 100pg | 1min | 1uM | others to 16 | 72C, 3min | 1.6ng X 2 | TSO.id02 | N2.id02 |
X2 | 1ng | 1min | 1uM | id01-02 | 10C,15min | 1uM | ||
X2 | NTC | 1min | 1uM | id07-08 | 10C,15min | 3.2ng | TSO.r02 | N2.id03 |
X12 | 100pg | 1min | 1uM | others to 16 | 10C,15min | 1.6ng X 2 | TSO.id04 | N2.id04 |
X2 | 100pg | 1min | 0.1uM | id01-02 | 72C, 3min | 1uM | ||
X2 | NTC | 1min | 0.1uM | id07-08 | 72C, 3min | 320pg | TSO.r02 | N2.id05 |
X12 | 10pg | 1min | 0.1uM | others to 16 | 72C, 3min | 0.16ng X 2 | TSO.id06 | N2.id06 |
X2 | 100pg | 1min | 0.1uM | id01-02 | 10C,15min | 1uM | ||
X2 | NTC | 1min | 0.1uM | id07-08 | 10C,15min | 320pg | TSO.r02 | N2.id07 |
X12 | 10pg | 1min | 0.1uM | others to 16 | 10C,15min | 0.16ng X 2 | TSO.id08 | N2.id08 |
Results
File:12.17.12 1-4.jpgFile:12.17.12 5-8.jpg
Size selection
- 100pg: >300, <300
- 10pg: >300, <300
- EtOH precipitation, resuspend in 11ul, 1ul for Qubit
#1.10pg,<300bp 2.60ng/ul 26ng #2.10pg,>300bp 1.45ng/ul 14.5ng #3.100pg,<300bp 10.7ng/ul 107ng #4.100pg,>300bp 7.41ng/ul 74.1ng
- Option1: mix <300bp and >300bp )(10pg and 100pg) for DSN
- Option2: reAmp for a few cycles individually
- Take option 2, reAmp #1 and #2 for 3 cycles (goes with 4 cycles for safe), #3 and #4 for 1 cycle