AlanFung:LabNotes/ASE/2010/2010-7-26
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RNA allelotyping on hybrids[edit]
Exp. #1 Perform CES36k capturing experiment on double stranded cDNAs[edit]
- Samples:
*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.
- Forward primers:
AmpF6.3Sol: AATGATACGGCGACCACCGACACTCTCAGATGTTATCGAGGTCCGAC
- Reverse primers:
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 | ||
- Size Selection