AlanFung:LabNotes/Capturing/2012-8-18: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 462: | Line 462: | ||
*Qubit quantification | *Qubit quantification | ||
==Pool library 1 | * 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== | |||
==Pool library Gel 2== | |||
==Pool library Gel 3== | |||
==Pool Library 11-25== | ==Pool Library 11-25== |
Revision as of 22:24, 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.
- 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
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