AlanFung:LabNotes/Capturing/2012-8-18: Difference between revisions

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==Indexing PCR with Kapa SYBR Fast==
==Indexing PCR with Kapa SYBR Fast==
*The 3 samples highlighted in red will use 15ul from the 1st round PCR (purified product) as the 2nd round PCR is already amplified with Kapa HiFi.
*First add Index primer
*Repeat PCR with same condition as first round of captured library in order to be consistance with the protocol
*Repeat PCR with same condition as first round of captured library in order to be consistance with the protocol
*Qiaquick column purify the 3 libraries that were taken out at the early stage of 1st round amplification
*Qiaquick column purify the 3 libraries that were taken out at the early stage of 1st round amplification
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*The 3 samples highlighted in red will use 15ul from the 1st round PCR (purified product) as the 2nd round PCR is already amplified with Kapa HiFi.
 
*First add Index primer
*Then add captured DNA
*Then add captured DNA
*Prepare Master Mix
*Prepare Master Mix

Revision as of 13:43, 18 August 2012

Indexing PCR with Kapa SYBR Fast

  • The 3 samples highlighted in red will use 15ul from the 1st round PCR (purified product) as the 2nd round PCR is already amplified with Kapa HiFi.
  • First add Index primer
  • Repeat PCR with same condition as first round of captured library in order to be consistance with the protocol
  • Qiaquick column purify the 3 libraries that were taken out at the early stage of 1st round amplification
  • Elute with 30ul EB, use 15ul for amplification
Content (50ul Capture) 9 rxn Volume Master Mix ChIPK36me3 Index ChIPK27me3 Index    
Captured DNA 27 Do not add to MM GM12878 1 GM12878 10    
2X Kapa SYBG qPCR master mix 50 495 H0 2 H0 11    
10uM AmpF6.3Sol 4 39.6 H7 3 H7 12    
10uM AmpR6.3Ind 4 Do not add to MM H14 4 H14 13    
H2O 15 148.5 H16 9 H16 14    
                 
                 
Content (20ul Capture) 13rxn Volume Master Mix WCE Index cDNA Index gDNA Index
Captured DNA 11 Do not add to MM GM12878 15 GM 22 H7 27
2X Kapa SYBG qPCR master mix 50 715 P10 16 H0 23 H14 28
10uM AmpF6.3Sol 4 57.2 DF2 17 H7 24 H16 29
10uM AmpR6.3Ind 4 Do not add to MM H0 18 H14 25    
H2O 31 443.3 H7 19 H16 26    
      H14 20        
      H16 21        
                 
Content (15ul Capture) 3rxn Volume Master Mix Type Sample Index      
Captured DNA 15 Do not add to MM ChIPK36me3 H0 2      
2X Kapa SYBG qPCR master mix 50 165 WCE DF2 17      
10uM AmpF6.3Sol 4 13.2 cDNA GM 22      
10uM AmpR6.3Ind 4 Do not add to MM            
H2O 27 89.1            
  • Then add captured DNA
  • Prepare Master Mix
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.
  • Master mix for 50ul reaction was frozen on the ice block, I realized this after adding in the MM to wells, ran pcr nothing amplified based on the PCR curve.
  • Qiaquick elute with 29ul
  • Redo amplification
Content (50ul Capture) 10 rxn Volume Master Mix ChIPK36me3 Index ChIPK27me3 Index
Captured DNA 27 Do not add to MM GM12878 1 GM12878 10
2X Kapa SYBG qPCR master mix 50 550 H0 2 H0 11
10uM AmpF6.3Sol 4 44 H7 3 H7 12
10uM AmpR6.3Ind 4 Do not add to MM H14 4 H14 13
H2O 15 165 H16 9 H16 14

Purification & Quantification

  • Take 1ul to runa TBE PAGE gel

File:ZhangLab 2 2012-08-18 05hr 02min.jpg File:ZhangLab 2 2012-08-18 05hr 00min.jpg File:ZhangLab 2 2012-08-18 04hr 58min.jpg

  • Qiaquick purify all libraries, elute with 30ul EB
  • Qubit quantification

Pool library 1-10

Pool Library 11-25