AlanFung:LabNotes/Capturing/2012-8-18

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Indexing PCR with Kapa SYBR Fast[edit]

  • 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.
  • 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            
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 (sample 1-10) 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[edit]

Sample # ID Sample
1 ChIPK36me3 GM12878
2 ChIPK36me3 H0
3 ChIPK36me3 H7
4 ChIPK36me3 H14
5 ChIPK36me3 H16
6 ChIPK27me3 GM12878
7 ChIPK27me3 H0
8 ChIPK27me3 H7
9 ChIPK27me3 H14
10 ChIPK27me3 H16
11 WCE GM12878
12 WCE P10
13 WCE DF2
14 WCE H0
15 WCE H7
16 WCE H14
17 WCE H16
18 cDNA GM
19 cDNA H0
20 cDNA H7
21 cDNA H14
22 cDNA H16
23 gDNA 27
24 gDNA 28
25 gDNA 29
  • 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
  • The gel quantification is giving me negative values for a lot of the libraries
  • I will just mix libraries based on their intensities at 180bp, and pool them based on the gel allocation, so we will have 3 set of libraries

Pool library Gel 1 (9 Libraries)[edit]

Sample ng Intensity Volume ul Total Intensity
100bp 1.25 12386 N/A N/A
200bp 2.5 15800 N/A N/A
400bp 5 19675 N/A N/A
1 3.21 16508 3.75 61942
2 27.28 61942 1.00 61942
3 7.28 24180 2.56 61942
4 2.74 15627 3.96 61942
5 3.71 17455 3.55 61942
6 2.56 15279 4.05 61942
7 2.98 16079 3.85 61942
8 3.53 17109 3.62 61942
9 7480 8.28 61942

Pool library Gel 2[edit]

Sample ng Intensity Volume ul Total Intensity
100bp 1.25 6552 N/A N/A
200bp 2.50 9857 N/A N/A
400bp 5.00 11732 N/A N/A
10 6.32 13777 1.49 20465
11 3493 5.86 20465
12 3217 6.36 20465
13 2.27 8539 2.40 20465
14 4205 4.87 20465
15 5110 4.00 20465
16 3254 6.29 20465
17 N/A N/A N/A N/A
18 11.50 20465 1.00 20465
#17 amplified w Kapa HiFi 5.38 12364.83 1.66 20465

Pool library Gel 3[edit]

Sample ng Intensity Volume ul Total Intensity
100bp 1.25 4274 1.43 6091
200bp 2.50 6091 1.00 6091
400bp 5.00 5827 1.05 6091
19 1738 3.51 6091
20 1619 3.76 6091
21 1665 3.66 6091
22 1994 3.05 6091
23 0.06 2545 2.39 6091
24 1517 4.02 6091
25 1616 3.77 6091

Nanodrop[edit]

  • Gel 1 Pool #1-9 27.8ng/ul
  • Gel 2 Pool #10-18 18.3ng/ul
  • Gel 3 Pool #19-25 12.3ng/ul

2% E-Gel Size Selection[edit]

  • Load 25ul pooled libraries for each well
  • 6ul H2O + 1ul LM ladder
  • Fill remaining wells w 30ul EB buffer, add 10ul during run
  • Collect libraries at 180BP
  • Pool 1 (sample 1-9) showed up as a smear with size around 100bp, this doesn't look right.
  • I will capture the libraries at multiple time point and rerun a TBE gel to verify the size

File:ZhangLab 2 2012-08-18 18hr 11min.jpg

  • 1-9_2, 10-18_1 and 19-25_1 are at the right size
  • Move on with qubit quantification and we can start the cluster generation