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

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Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit
Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit


                            A       B     C    
                          64       65     72    73    75    
   RNA                    6ul    2.5ul  10ul
   RNA                    3ul      3ul    3ul  3ul    4ul
   10X DNase I buffer      5ul      5ul  5ul
   10X DNase I buffer      5ul      5ul    5ul  5ul    5ul
   RNase-Free DNase I      2.5ul  2.5ul  2.5ul
   RNase-Free DNase I      2.5ul  2.5ul  2.5ul  2.5    2.5
   H2O                   36.5ul     40ul 32.5ul
   H2O                   39.5ul  39.5ul  32.5ul 32.5  31.5
   37C 10min  
   37C 10min  


Line 35: Line 35:
Reverse transcription
Reverse transcription


                   A      B      C      
                   64    65      72    73    75      
  RNA            8ul    8ul    8ul     
  RNA            8ul    8ul    8ul     
  10mM dNTP      1ul    1ul    1ul     
  10mM dNTP      1ul    1ul    1ul     

Revision as of 22:44, 2 July 2009

DNA and RNA extraction from samples and ASE validation for fuzzlmethylated genes

64: 1515.7ng/ul 2.1/2.20 65: 1515.9 2.11/2.19 72: 1638 2.1/2.18 73: 1416.4 2.11/2.14 75: 838.3 2.13/2.11



Making ds-cDNA for Hybrids/BJ

   * 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

                          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 
   * 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     65      72    73    75      
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.