Jie:LabNotes/CpgSeq/2009-7-2: Difference between revisions
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Revision as of 20:37, 4 July 2009
DNA and RNA extraction from samples and ASE validation for fuzzlmethylated genes
DNA and RNA extraction with Qiagen Allprep DNA/RNA Mini kit
sample | DNA concentration | DNA 260/280;260/230 | RNA concentration | RNA 260/280;260/230 |
GM12864 | 94.1ng/ul x 100ul | 1.97/0.17 | 1515.7ng/ul x 30ul | 2.1/2.2 |
GM12865 | 130.9ng/ul/ul x 100ul | 1.98/1.44 | 1515.9ng/ul x 30ul | 2.11/2.19 |
GM12872 | 217.8ng/ul x 100ul | 1.94/1.83 | 1638ng/ul x 30ul | 2.1/2.18 |
GM12873 | 82.6ng/ul x 100ul | 1.92/1.49 | 1416.4ng/ul x 30ul | 2.11/2.14 |
GM12875 | 140ng/ul x 100ul | 1.89/1.03 | 838.3ng/ul x 30ul | 2.13/2.11 |
Making cDNA
64 65 72 73 75 RNA 3ul 3ul 3ul 3ul 4ul 10X DNase I buffer 5ul 5ul 5ul 5ul 5ul RNase-Free DNase I 2.5ul 2.5ul 2.5ul 2.5 2.5 H2O 39.5ul 39.5ul 32.5ul 32.5 31.5 37C 10min Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit * 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
64~75 RNA 8ul 10mM dNTP 1ul 50uM dT12-18 1ul 65C 5min -> chill on ice for 1min Add 10X RT buffer 2ul 25mM MgCl2 4ul 0.1M DTT 2ul RNaseOUT 1ul Superscript III 1ul Incubate all tube at 50C for 50min.
Cleanup the first-strand reactions with qiaquick columns. quantify: GM12864: 74.8ng/ul; 2.17/1.16; GM12865: 72.3ng/ul; 2.16/1.09; GM12872: 79.1ng/ul; 2.08/0.78; GM12873: 76.4ng/ul; 2.16/1.17; GM12875: 66.4ng/ul; 2.17/1.14;
PCR to validat the ASE
The primers and PCR conditions had been optimized on 09-06-29. refer to LabNotes on [1] x 40 2x iQ supermix 20 800 Primer F (25uM) 0.5 0.5 each Primer R (25uM) 0.5 0.5 each cDNA samples as above: 0.1 dH2O 20 800 total 40ul
For 12864/12872/12873: A1, C1-E1, A3-C3, E3-F3, H3, A5. For 12865/12875: G1, H1, D3. The annealing temperature for A5 and H1 is 58C. Annealing Temperature for others are 62C. 96C 3min-> 37x (95C 30sec, 58C or 62C 1min, 72C 1min)-> 72C 5min.
File:20090704 ASE valid 1.jpg20090704_ASE_valid_1
File:20090704 ASE valid 2.jpg20090704_ASE_valid_2