Revision as of 23:54, 15 July 2012 by >Alan6017518
Library Construction of Dr. Yi Zhang's DiP libraries Sample
Samples Preparation Protocol from Li
File:0712 DIP-seq samples.pdf
- Received samples from Li on Jul 13 2012, 12 samples total in a yellow box, transfer to -20C
qPCR with N2 Barcodes
DIP Sample
|
N2 Barcode
|
shCONT-input
|
1
|
shCONT-IgG
|
2
|
shCONT-5mC
|
3
|
shCONT-5hmC
|
4
|
shCONT-5fc
|
5
|
shCONT-5caC
|
6
|
shTDG-input
|
7
|
shTDG-IgG
|
8
|
shTDG-5mC
|
9
|
shTDG-5hmC
|
10
|
shTDG-5fC
|
11
|
shTDG-caC
|
12
|
Content
|
Volume Per Reaction
|
MM
|
Template
|
10
|
Do not add to MM
|
5uM PCR F+R Barcode
|
1
|
Do not add to MM
|
50X SYBG
|
0.2
|
2.64
|
2X PhusionHF MM
|
25
|
330
|
H20
|
13.8
|
182.16
|
Total Volume
|
50
|
660
|
- Add 20ul 10uM N2-bar-code primers into strip tube
- Add 20ul 10uM PCR_F primers and mix
- We have 5uM F+R N2 Ind primer (Store at 4C Alice yellow 0.2ul tube rack)
- Add 10ul template (for low volume sample add 5ul sample and add 5ul h2o) and 1ul barcode primers (using multi channel pipette) to strip tube
- Add 39ul MM to each well
98C 30sec
(98C 10sec -> 63C 20sec-> 72C 30sec) x 20 cycles
72C 2min
15C hold.
Gel quant
Qubit
DIP Sample
|
N2 Barcode
|
Sample Concentration ng/ul
|
Volume ul
|
Amount ng
|
shCONT-input
|
1
|
3.62
|
30
|
108.6
|
shCONT-IgG
|
2
|
2.36
|
30
|
70.8
|
shCONT-5mC
|
3
|
2.64
|
30
|
79.2
|
shCONT-5hmC
|
4
|
3.54
|
30
|
106.2
|
shCONT-5fc
|
5
|
3.58
|
30
|
107.4
|
shCONT-5caC
|
6
|
3.82
|
30
|
114.6
|
shTDG-input
|
7
|
3.54
|
30
|
106.2
|
shTDG-IgG
|
8
|
2.4
|
30
|
72
|
shTDG-5mC
|
9
|
3.06
|
30
|
91.8
|
shTDG-5hmC
|
10
|
4.04
|
30
|
121.2
|
shTDG-5fC
|
11
|
3.9
|
30
|
117
|
shTDG-caC
|
12
|
3.18
|
30
|
95.4
|