Jie:LabNotes/ASE/2009-6-10
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Making ds-cDNA for Hybrids/BJ[edit]
* Total RNA Samples: o A: Hybrid1-ES: 750ng/ul o B: Hybrid1-ES: 1.9ug/ul o C: BJ: 520ng/ul
Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit
A B C RNA 6ul 2.5ul 10ul 10X DNase I buffer 5ul 5ul 5ul RNase-Free DNase I 2.5ul 2.5ul 2.5ul H2O 36.5ul 40ul 32.5ul 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;
Reverse transcription
A B C RNA 8ul 8ul 8ul 10mM dNTP 1ul 1ul 1ul 50uM dT12-18 1ul 1ul 1ul 65C 5min -> chill on ice for 1min Add 10X RT buffer 2ul 2ul 2ul 25mM MgCl2 4ul 4ul 4ul 0.1M DTT 2ul 2ul 2ul RNaseOUT 1ul 1ul 1ul Superscript III 1ul 1ul 1ul Incubate all tube at 50C for 50min.
2nd strand synthesis
Cleanup the first-strand reactions with qiaquick columns.
To the 30ul reactions, add 10ul NEBuffer 2 5ul 10mM dNTP 2.5ul DNA Pol I(10U/ul) 1ul RNaseH 1ul H2O 10.5ul Incubate at 16C for 2h, purified with Qiaquick columns.
Yield (use DNA's absorbance): RNA A: Hybrid1-ES(750ng/ul): 22.9ng/ul x 30ul 18.1ng/ul B: Hybrid1-ES(1.9ug/ul): 80.2ng/ul x 30ul 63.4ng/ul C: BJ(520ng/ul): 94.4ng/ul x 30ul 72.3ng/ul
capture with CES27k9bp[edit]
B C Sample 8ul 8ul 10x buffer 2ul 2ul CES27k9bp 5ul 5ul H2O 5ul 5ul
SLN mixture:
ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul Stoffel;0.5U/ulLigase;0.5uM dNTP 0.1ul 0.1ul 0.1ul 0.2ul (1uM)0.5ul x20 2ul 2ul 2ul 4ul 10ul
95C 2min -> 60C 24h -> add 2ul SLN mix (dNTP 0.5uM, Stoffel 2U/ul, AmpLigase 1U/ul) -> 60C 18h -> 95C 5min -> 37c 1min -> add 2ul Exo I&III -> 37C 1h -> 94C 5min -> 4C hold
PCR[edit]
Template 10ul x4 2X iProof Mastermix 50ul 200ul AmpF6.2SoL (10uM) 4ul 16ul AmpR6.2SoL (10uM) 4ul 16ul 50X SYBG I 0.4ul 1.6ul H2O 31.6ul 126.4ul
I did 20 cycles for BJ and 21 cycles for Hybrid_1.
File:20090615 CES27k9bp Hybrid1 BJ.jpg20090615_CES27k9bp_Hybrid1_BJ
Qiaquick purification and gel quantification:
Hybrid_1: 8.6ng/ul x 30ul BJ: 14.3ng/ul x 30ul
I send the two samples to Zhen Ye as follows:
Hybrid_1: 4.3ng/ul (37nM) x 5ul BJ: 7.2ng/ul (62nM) x 5ul