Rui:LabNotes/SingleCell/2012-12-17: Difference between revisions

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* 10pg: >300, <300
* 10pg: >300, <300
* EtOH precipitation, resuspend in 11ul, 1ul for Qubit
* EtOH precipitation, resuspend in 11ul, 1ul for Qubit
  10pg,<300 2.60ng/ul    26ng
  #1.10pg,<300bp         2.60ng/ul    26ng
  10pg,>300 1.45ng/ul    14.5ng
  #2.10pg,>300bp         1.45ng/ul    14.5ng
  100pg,<300 10.7ng/ul    107ng
  #3.100pg,<300bp 10.7ng/ul    107ng
  100pg,>300 7.41ng/ul    74.1ng
  #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, #3 and #4 for 1 cycle
 
#1 and #2 goes with 4 cycles for safe [[File:12.18.12_reAmp.jpg|400px]]

Revision as of 03:22, 19 December 2012

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

File:12.17.12 gel.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, #3 and #4 for 1 cycle
  1. 1 and #2 goes with 4 cycles for safe File:12.18.12 reAmp.jpg