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==RNA allelotyping on hybrids== ===Exp. #1 Perform CES36k capturing experiment on double stranded cDNAs=== *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. ---- {| {{table}} | 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 {| {{table}} | ||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|300px]] **E-gel size Selection for BJ [[File:ZhangLab_2 2010-07-30 16hr 03min.jpg|300px]] *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 {| {{table}} | 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 {| {{table}} | ||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
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