Revision as of 18:40, 20 August 2010 by >Alan6017518
RNA allelotyping on hybrids[edit]
Exp. #1 Perform CES36k capturing experiment on double stranded cDNAs[edit]
*Samples:
**A: 1000-somatic: 61.9ng/ul x 15ul
**B: 1000-iPS: 87.8ng/ul x 15ul
**C: 1034-iPS: 53.5ng/ul x 15ul
**D: A2: 49.7ng/ul x 15ul
**E: B4: 44.8ng/ul x 15ul
**F: 1016-somatic: 58.1ng/ul x 15ul
**G: 1016-iPS: 60.6ng/ul x 15ul
**H: B5-iPS: 55.5ng/ul x 15ul
**BJ: 27.2ng/ul x 15ul
**HUES6: 40.9ng/ul x 15ul
- Probes: CES36k18bp, 590nM (prepared by Kun on 7/16/2010)
- Target:Probe ratio=200. With 200ng cDNA template, we need 1.4ul of probes.
Sample |
ng/ul |
Template |
Probes |
10X Buffer |
H2O
|
A |
61.9 |
3.23 |
1.4 |
1 |
4.37
|
B |
87.8 |
2.28 |
1.4 |
1 |
5.32
|
C |
53.5 |
3.74 |
1.4 |
1 |
3.86
|
D |
49.7 |
4.02 |
1.4 |
1 |
3.58
|
E |
44.8 |
4.46 |
1.4 |
1 |
3.14
|
F |
58.1 |
3.44 |
1.4 |
1 |
4.16
|
G |
60.6 |
3.3 |
1.4 |
1 |
4.3
|
H |
55.5 |
3.6 |
1.4 |
1 |
4
|
BJ |
27.2 |
7.35 |
1.4 |
1 |
0.25
|
HUES6 |
40.9 |
4.89 |
1.4 |
1 |
2.71
|
|
- Add in two drops of mineral oil using P200 pipette
- Spin down
Saved under Kun KZ-S-MIP
95c 30sec -> cool down to 60C at 0.1C/sec -> 60C 20h
-> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP)
-> 60C 20h-> 94C 2min -> add 1ul Exo I/III mix-> 37C 1h -> 94C 2min -> 4C hold.
PCR
2X iProof Mastermix 50ul
AmpF6.3NH2 (100uM) 0.2ul
AmpR6.3NH2 (100uM) 0.2ul
50X SYBG I 0.4ul
H2O 40ul
Captured DNA 10ul
|
X11
|
2X iProof Mastermix |
550
|
AmpF6.3NH2 (100uM) |
2.2
|
AmpR6.3NH2 (100uM) |
2.2
|
50X SYBG I |
4.4
|
h2o |
440
|
|
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 20
Monitoring the reactions and terminate the program right before the amplification curves reach the plateau.
Purified with Qiaquick columns, quantified with Nanodrop:
(A) 6.1ng/ul x 40ul
(B) 9.3ng/ul x 40ul
(C) 8.4ng/ul x 40ul
(D) 7.7ng/ul x 40ul
(E) 7.4ng/ul x 40ul
(F) 6.0ng/ul x 40ul
(G) 4.6ng/ul x 40ul
(H) 6.1ng/ul x 40ul
(Hues6)6.4ng/ul x 40ul
(BJ)13.5ng/ul x 40ul
File:ZhangLab 2 2010-07-30 12hr 45min.jpg
- E-gel size Selection for BJ
File:ZhangLab 2 2010-07-30 16hr 03min.jpg
- Set up 2nd round PCR to add barcodes.
AmpF6.3Sol: AATGATACGGCGACCACCGACACTCTCAGATGTTATCGAGGTCCGAC
Sample ID Primer ID Bardcode (rev. comp.)
(A) AmpR6.3Ind1
(B) AmpR6.3Ind2
(C) AmpR6.3Ind3
(D) AmpR6.3Ind4
(E) AmpR6.3Ind5
(F) AmpR6.3Ind6
(G) AmpR6.3Ind7
(H) AmpR6.3Ind8
(Hues6) AmpR6.3Ind9
(BJ) AmpR6.3Ind10
PCR x 8
Template: 4ul
2x Kapa SYBG qPCR Master Mix: 50ul
100uM AmpF6.3Sol: 0.4ul
100uM AmpR6.3IndXX: 0.4ul
H2O 46ul
PCR |
1 |
X11
|
2X Kapa SYBG qPCR Master Mix |
50 |
550
|
100uM AmpF6.3Sol |
0.4 |
4.4
|
H20 |
46 |
506
|
|
95C 30sec -> (95C 3sec -> 60C 20sec) x 5 -> 60C 1min
Purified with Qiaquick columns, quantified with Nanodrop, and pooled in equal ratios.
Sample
A: 17.4ng/ul x 40ul
B: 28.3ng/ul x 40ul
C: 24.7ng/ul x 40ul
D: 22.1ng/ul x 40ul
E: 21.2ng/ul x 40ul
F: 20.2ng/ul x 40ul
G: 16.7ng/ul x 40ul
H: 16.9ng/ul x 40ul
Hues6: 20.7ng/ul x 40ul
BJ: 17.9ng/ul x 40ul
|
ng/ul |
To get 100ng (ul) |
|
A |
17.4 |
5.75 |
|
B |
28.3 |
3.53 |
|
C |
24.7 |
4.05 |
|
D |
22.1 |
4.52 |
|
E |
21.1 |
4.74 |
|
F |
20.2 |
4.95 |
|
G |
16.7 |
5.99 |
|
H |
16.9 |
5.92 |
|
Hues6 |
20.7 |
4.83 |
|
BJ |
17.9 |
5.59 |
|
Total Volume |
|
49.87 |
|
Concentration |
|
|
20ng/ul
|
|