AlanFung:LabNotes/Capturing/2012-6-26

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  • New sample list (some of the older concentration are just for calculations, the latest sample list ignored those)
  • H7 and H14 were captured based on the wrong concentration, we used much less sample than expected

Always up to date sample list

Capturing Reaction[edit]

  • Add 2 drops of mineral oil to prevent evaporation
ChIPK36me3 Sample Vol Vol of probes 10X Ampligase Buffer H2O Total
P10 40 0.6 5.00 4.40 50.00
DF2 40 0.6 5.00 4.40 50.00
ChIPK27me3 Sample Vol Vol of probes 10X Ampligase Buffer H2O Total
P10 40.00 0.65 5.00 4.35 50.00
DF2 40.00 1.00 5.00 4.00 50.00
cDNA Sample Vol Vol of probes 10X Ampligase Buffer H2O Total
P10E3 5.25 2.49 2.00 10.26 20.00
DF2 5.25 2.49 2.00 10.26 20.00
gDNA Sample Vol Vol of probes 10X Ampligase Buffer H2O Total
GM 5.25 2.49 2.00 10.26 20.00
P10E3 5.25 2.49 2.00 10.26 20.00
DF2 5.25 2.49 2.00 10.26 20.00
H0 5.25 2.49 2.00 10.26 20.00
GmgDNA:H0gDNA::1:1    5.25 2.49 2.00 10.26 20.00

Prepare SLN Mix[edit]

  • Dilute 1mM dNTP 1 in 100 to 10uM dNTP
SLN Mix for 20ul Rxn 1x 7.7x
dNTP (10uM) 0.5 3.85
Stoffel (10U/ul) 0.2 1.54
Ampligase (5U/ul) 0.2 1.54
H2O 0.1 0.77
     
SLN Mix for 50ul Rxn 1x 4.4x
dNTP (10uM) 1.25 5.5
Stoffel (10U/ul) 0.5 2.2
Ampligase (5U/ul) 0.5 2.2
H2O 0.25 1.1

Prepare Exo I and III Mix[edit]

Exo I & III for 20ul Rxn 1X 7.7x
Exo I (20U/ul) 1 7.7
ExoIII (200U/ul) 1 7.7
     
Exo I & III for 50ul Rxn 1X 4.4x
Exo I (20U/ul) 2.5 11
ExoIII (200U/ul) 2.5 11
  • When adding regents to the tube make sure you penetrate the oil layer
95C 2min -> -0.2C/sec to 60C -> 60C 24h 
   -> add 1ul/2.5ul(for 50ul rxn) SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) 
   -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul(5ul for 50ul rxn) Exo I&III
   -> 37C 2h -> 90C 5min -> 4C hold

Indexing qPCR[edit]

  • Perform one step qPCR using Indexing seq. primer
Content (50ul Capture) 4 rxn Volume Master Mix ChIPK36me3 IND
Captured DNA 27 Do not add to MM P10 Round 2 3
2X Kapa SYBG qPCR master mix 50 220 DF2 Round 2 4
10uM AmpF6.3Sol 4 17.6  
10uM AmpR6.3Ind 4 Do not add to MM ChIPK27me3 IND
H2O 15 66 P10 Round 2 5
      DF2 Round 2 6
Content (20ul Capture) 7rxn Volume Master Mix  
Captured DNA 11 Do not add to MM cDNA IND
2X Kapa SYBG qPCR master mix 50 385 P10E3 7
10uM AmpF6.3Sol 4 30.8 DF2 8
10uM AmpR6.3Ind 4 Do not add to MM  
H2O 31 238.7 gDNA IND
      GM 9
      P10E3 10
      DF2 11
      H0 12
      GmgDNA:H0gDNA::1:1    13
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40
Monitor the reactions and terminate the program right before the amplification curves reach the plateau.

File:ZhangLab 2 2012-07-06 15hr 58min.jpg

  • after talking to Kun, it appears that the larger amplicons are 2 circles forming when the polymerase for some reason didn't fall off and continue the amplification.
  • Quantify all amplicons with qubit and pool to perform a size selection
  • Qiaquick all samples elute with 30ul EB
gDNA ng/ul To get 180ng
H7 7.24 13.81
H14 6.16 16.23
   
ChIPK36me3 ng/ul To get 180ng
P10 14.42 6.93
DF2 16.08 6.22
 
ChIPK27me3 ng/ul To get 180ng
P10 10.92 9.16
DF2 16.52 6.05
 
cDNA ng/ul To get 180ng
P10E3 5.98 16.72
DF2 4.54 22.03
 
gDNA ng/ul To get 180ng
GM 9.72 10.29
P10E3 7.62 13.12
DF2 9.44 10.59
H0 8.28 12.08
GmgDNA:H0gDNA::1:1    12.5 8.00
  • 15.47ng/ul after combine

Gel Size Selection[edit]

  • 200ng/well is the limite for 5 well gel
  • I will put 13ul of the pooled library ~200ng into each well