Jie:LabNotes/ASE/2009-6-10
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Making ds-cDNA for Hues6/Hybrids/BJ
* Total RNA Samples: o A: Hues6-ES: 600ng/ul o B: Hues6-EB: 1ug/ul o C: Hybrid1-ES: 750ng/ul o D: Hybrid1-EB: 170ng/ul o E: Hybrid2-ES: 1.5ug/ul o F: Hybrid2-EB: 48ng/ul o G: BJ: 110ng/ul o H: Hybrid1-ES (diluted): 380ng/ul
Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit
A B C D E F G RNA 8ul 5ul 6ul 30ul 3ul 80ul 20ul 10X DNase I buffer 5ul 5ul 5ul 5ul 5ul 10ul 5ul RNase-Free DNase I 2ul 2ul 2ul 2ul 2ul 4ul 2ul H2O 35ul 38ul 37ul 13ul 40ul 6ul 3ul 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
H was not DNase treated.
A B C D E F G H RNA 8ul 8ul 8ul 8ul 8ul 8ul 8ul 8ul 10mM dNTP 1ul 1ul 1ul 1ul 1ul 1ul 1ul 1ul 50uM dT12-18 1ul 1ul 1ul 1ul 1ul 1ul 1ul 1ul 65C 5min -> chill on ice for 1min Add 10X RT buffer 2ul 2ul 2ul 2ul 2ul 2ul 2ul 2ul 25mM MgCl2 4ul 4ul 4ul 4ul 4ul 4ul 4ul 4ul 0.1M DTT 2ul 2ul 2ul 2ul 2ul 2ul 2ul 2ul RNaseOUT 1ul 1ul 1ul 1ul 1ul 1ul 1ul 1ul Superscript III 1ul 1ul 1ul 1ul 1ul 1ul 1ul 1ul Incubate all tube at 50C for 50min.
[edit] 2nd strand synthesis
Cleanup the first-strand reactions with G-25 columns.
To the ~40ul reactions, add 10ul NEBuffer 2 10ul 10mM dNTP 5ul DNA Pol I(10U/ul) 2ul RNaseH 2ul H2O adjust to 100ul Incubate at 16C for 2h, purified with Qiaquick columns. Yield (use RNA's absorbance): A: Hues6-ES: 50ng/ul x 30ul B: Hues6-EB: 55ng/ul x 30ul C: Hybrid1-ES: 30ng/ul x 30ul D: Hybrid1-EB: 63ng/ul x 30ul E: Hybrid2-ES: 58ng/ul x 30ul F: Hybrid2-EB: 53ng/ul x 30ul G: BJ: 44ng/ul x 30ul H: Hybrid1-ES: 56ng/ul x 30ul