Jie:LabNotes/CpgSeq/2009-7-2

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Revision as of 23:33, 2 July 2009 by >Sam Chiang
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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.


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.