AlanFung:LabNotes/Capturing/2012-8-10

From ZhangLabWiki
Revision as of 07:19, 18 August 2012 by >Alan6017518 (→‎Indexing PCR with Kapa SYBR Fast)
Jump to navigation Jump to search

Capturing Remaining Gimelbrant lab Sample

  • Add 2 drops of mineral oil to prevent evaporation
  • Samples stored at tall -20C Alan Rack white box red label "Gimelbrant Lab"
ChIPK36me3 Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM12878 0.90 40.00 0.45 2 7.6 50
H0 1.00 40.00 0.50 2 7.5 50
H7 0.60 40.00 0.30 2 7.7 50
H14 0.50 40.00 0.25 2 7.8 50
H16 0.60 40.00 0.30 2 7.7 50
                
ChIPK27me3 Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM12878 1.20 40.00 0.60 2 7.4 50
H0 2.4 40.00 1.20 2 6.8 50
H7 1.2 40.00 0.60 2 7.4 50
H14 0.5 40.00 0.25 2 7.8 50
H16 1.3 40.00 0.65 2 7.4 50
                
WCE Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM12878 35.39 5.65 2.49 2 9.9 20
P10 35.39 5.65 2.49 2 9.9 20
DF2 35.39 5.65 2.49 2 9.9 20
H0 35.39 5.65 2.49 2 9.9 20
H7 35.39 5.65 2.49 2 9.9 20
H14 35.39 5.65 2.49 2 9.9 20
H16 35.39 5.65 2.49 2 9.9 20
                
cDNA Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM 38.12 5.25 2.49 2 10.3 20
H0 38.12 5.25 2.49 2 10.3 20
H7 38.12 5.25 2.49 2 10.3 20
H14 38.12 5.25 2.49 2 10.3 20
H16 38.12 5.25 2.49 2 10.3 20
                
gDNA Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
H7 38.12 5.25 2.49 2 10.3 20
H14 38.12 5.25 2.49 2 10.3 20
H16 38.12 5.25 2.49 2 10.3 20
  • When adding regents to the tube make sure you penetrate the oil layer
Store under βCES36K
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

Prepare SLN Mix

  • Dilute 1mM dNTP 1 in 100 to 10uM dNTP
SLN Mix for 20ul Rxn 1x 16x
dNTP (10uM) 0.5 8
Stoffel (10U/ul) 0.2 3.2
Ampligase (5U/ul) 0.2 3.2
H2O 0.1 1.6
     
SLN Mix for 50ul Rxn 1x 11x
dNTP (10uM) 1.25 13.75
Stoffel (10U/ul) 0.5 5.5
Ampligase (5U/ul) 0.5 5.5
H2O 0.25 2.75

Prepare Exo I and Exo III Mix

Exo I & III for 20ul Rxn 1X 16x
Exo I (20U/ul) 1 16
ExoIII (200U/ul) 1 16
     
Exo I & III for 50ul Rxn 1X 11x
Exo I (20U/ul) 2.5 6.25
ExoIII (200U/ul) 2.5 6.25

Indexing qPCR

  • Perform one step qPCR using Indexing seq. primer
Content (50ul Capture) 4 rxn Volume Master Mix ChIPK36me3 Index ChIPK27me3 Index    
Captured DNA 27 Do not add to MM GM12878 1 GM12878 10    
2X Kapa HiFi 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    
SYBR Green 50X 0.4 4.4            
                 
Content (20ul Capture) 7rxn 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 50 825 P10 16 H0 23 H14 28
10uM AmpF6.3Sol 4 66 DF2 17 H7 24 H16 29
10uM AmpR6.3Ind 4 Do not add to MM H0 18 H14 25    
H2O 31 511.5 H7 19 H16 26    
SYBR Green 50X 0.4 6.6 H14 20        
      H16 21        
95C 2min -> (98C 20S -> 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.

Indexing PCR for 3 samples

Indexing PCR with Kapa SYBR Fast

  • Repeat PCR with same condition as first round of captured library in order to be consistance
  • Qiaquick column purify the 3 libraries that were taken out at the early stage of 1st round amplification
  • Elute with 30ul EB
Content (50ul Capture) 4 rxn Volume Master Mix ChIPK36me3 Index ChIPK27me3 Index    
Captured DNA 27 Do not add to MM GM12878 1 GM12878 10    
2X Kapa HiFi 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    
SYBR Green 50X 0.4 3.96            
                 
Content (20ul Capture) 7rxn 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    
SYBR Green 50X 0.4 5.72 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            
SYBR Green 50X 0.4 1.32            
  • 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
  • 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.

Purification & Quantification

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