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