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

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>Sam Chiang
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>Sam Chiang
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===Making cDNA ==
 
==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     
                           64        65    72    73    75     
Line 40: Line 30:
   H2O                    39.5ul  39.5ul  32.5ul 32.5  31.5
   H2O                    39.5ul  39.5ul  32.5ul 32.5  31.5
   37C 10min  
   37C 10min  
 
  Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit
     * Add 4 volumes of RNA binding buffer;
     * Add 4 volumes of RNA binding buffer;
     * Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s;
     * Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s;
Line 46: Line 36:
     * Add 10ul DNase/RNase-free water, spin @15k rpm for 30s;
     * Add 10ul DNase/RNase-free water, spin @15k rpm for 30s;


Reverse transcription
Reverse transcription


                   64     65      72    73    75       
                   64~75       
  RNA            8ul     8ul    8ul   
  RNA            8ul  
  10mM dNTP      1ul     1ul    1ul   
  10mM dNTP      1ul      
  50uM dT12-18    1ul     1ul    1ul   
  50uM dT12-18    1ul  
                
                
  65C 5min -> chill on ice for 1min
  65C 5min -> chill on ice for 1min
  Add   
  Add   
  10X RT buffer  2ul    2ul      2ul     
  10X RT buffer  2ul     
  25mM MgCl2     4ul    4ul     4ul     
  25mM MgCl2      4ul     
  0.1M DTT        2ul     2ul      2ul   
  0.1M DTT        2ul        
  RNaseOUT        1ul    1ul      1ul     
  RNaseOUT        1ul     
  Superscript III 1ul    1ul      1ul   
  Superscript III 1ul     
  Incubate all tube at 50C for 50min.
  Incubate all tube at 50C for 50min.


 
Cleanup the first-strand reactions with qiaquick columns.  
2nd strand synthesis
quantify:
 
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.

Revision as of 00:06, 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: