Jie:LabNotes/CpgSeq/2009-7-2: Difference between revisions

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  Cleanup the first-strand reactions with qiaquick columns.  
  Cleanup the first-strand reactions with qiaquick columns.  
  quantify:
  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;

Revision as of 01:13, 3 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;