AlanFung:LabNotes/ASE/2010/2010-8-16
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RNA allelotyping on hybrids[edit]
Exp. #3 Perform CES36k capturing experiment on ds-cDNAs[edit]
ds cDNA synthesis[edit]
*Samples: Nanodrop
- H1:Hybrid 1 p26: 1800ng/ul x 30ul 1393ng/ul
- 1034-somatic: Egg donor somatic: 100ng/ul x 130ul 100.1ng/ul
gdna[edit]
- H1: 240ng/ul x 100ul 175.5ng/ul
Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit
H1 | 1034s | |
RNA | 3 | 43 |
10X Dnase I Buffer | 5 | 5 |
Rnase-Free Dnase I | 2 | 2 |
H2O | 40 | 0 |
37C 10min Add 4 volumes of RNA binding buffer; Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s; Add 200ul RNA Wash buffer, spin @15k rpm for 30s; Add 10ul DNase/RNase-free water, spin @15k rpm for 30s; Yield: H1: 310.6ng/ul x 10ul 1034s: 336.4ng/ul x 10ul
Reverse transcription
H1 | 1034s | |
RNA | 8 | 8 |
10mM dNTP | 1 | 1 |
50uM dT12-18 | 1 | 1 |
65C 5min -> chill on ice for 1min Add H1 1034s 10X RT buffer 2 2 25mM MgC12 4 4 0.1M DTT 2 2 RnaseOUT 1 1 Superscript III 1 1 Incubate all tube at 50C for 50min.
2nd strand synthesis saved under Alan>2ss Add 5X 2nd strand buffer: 30ul 10mM dNTP 3ul DNA Pol I(10U/ul) 4ul RNase H(2U/ul) 1ul H2O 92ul 16C 2h -> 70C 10min -> add 2ul RNase I -> 37C 30min Purified with MinElute columns.
Yield: H1: 252.5ng/ul x 15ul 1034s: 285.8ng/ul x 15ul
Sample volume TBE Buffer 6X Loading Dye Total Volume /4
H1+gDNA 14.73 16 28 58.73 14.68 1034+cDNA 27.75 4 28 59.75 14.94
RNA allelotyping on hybrids[edit]
Exp. #1 Perform CES36k capturing experiment on double stranded cDNAs[edit]
- 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 H1 (cDNA) 252.5 0.79 1.4 1 6.81 1034s (cDNA) 285.8 0.7 1.4 1 6.9 H1 (gDNA) 175.5 1.14 1.4 1 6.46
- 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.
- Perform one step qPCR using Indexing seq. primer
PCR Captured DNA 1034s (cDNA) 10 2X Kapa SYBG qPCR Master Mix 50 100uM AmpF6.3Sol 0.4 100uM AmpR6.3Ind11 0.4 H2O 46 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40
Monitoring the reactions and terminate the program right before the amplification curves reach the plateau.
- Stop at cycle 12
File:ZhangLab 2 2010-08-19 12hr 33min.jpg
- One step PCR worked well, move on with the 2 remaining samples
- Perform one step qPCR using Indexing seq. primer
PCR Captured DNA 1034s (cDNA) 10 2X Kapa SYBG qPCR Master Mix 50 100uM AmpF6.3Sol 0.4 100uM AmpR6.3Ind* 0.4 H2O 46 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40
Monitoring the reactions and terminate the program right before the amplification curves reach the plateau. AmpR6.3Ind1 - H1 gDNA AmpR6.3Ind12 - H1 cDNA
- TBU Gel Quantification
Pre-run TBU gel 75C 7 min -> cooling rack 5 min -> Load gel
File:ZhangLab 2 2010-08-19 17hr 19min.jpg
Purified with Qiaquick columns, quantified with Nanodrop and pooled in equal ratios. H1(cDNA) 21.3ng/ul x 30ul H1(gDNA) 26.9ng/ul x 30ul 1034s 32.0ng/ul x 30ul Pool H1(gDNA) and H1(cDNA) in equal ratios ng/ul To get 100ng H1cDNA 21.3 4.69 H1gDNA 26.9 3.72
Pool H1 (gDNA/cDNA mix) with gDNA mix in 4:6 ratio Pool 1034s (cDNA) with cDNA mix in 2:8 ratio ng/ul Ng Needed Volume H1 mix 23.78 60 2.52 1034s 32 30 0.94 cDNA Mix 20 120 6 gDNA Mix 77.7 90 1.16
- Size Selection
Sample volume | TBE Buffer | 6X Loading Dye | Total Volume | /4 | |
H1+gDNA | 14.73 | 16 | 28 | 58.73 | 14.68 |
1034+cDNA | 27.75 | 4 | 28 | 59.75 | 14.94 |
Turn on centrifuge let it cool down to 4C
- Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol.
- Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
- Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
- Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
- Let dry in hood ~ 10 min.
- Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
Store at 4C if needed, spin down before proceeding
- Qubit Quantification
cDNA-2.42ng/ul (22.1nM) gDNA-1.19ng/ul (10.87nM)