Jie:LabNotes/ASE/2009-6-10: Difference between revisions

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H was not DNase treated.
H was not DNase treated.


                   A      B      C       D     E    F    G    H
                   A      B      C       
  RNA            8ul    8ul    8ul    8ul    8ul  8ul  8ul  8ul
  RNA            8ul    8ul    8ul     
  10mM dNTP      1ul    1ul    1ul    1ul    1ul  1ul  1ul  1ul
  10mM dNTP      1ul    1ul    1ul     
  50uM dT12-18    1ul    1ul    1ul    1ul    1ul  1ul  1ul  1ul
  50uM dT12-18    1ul    1ul    1ul     
                
                
  65C 5min -> chill on ice for 1min
  65C 5min -> chill on ice for 1min
  Add   
  Add   
  10X RT buffer  2ul    2ul      2ul    2ul  2ul    2ul      2ul    2ul
  10X RT buffer  2ul    2ul      2ul     
  25mM MgCl2      4ul    4ul      4ul    4ul  4ul    4ul      4ul    4ul
  25mM MgCl2      4ul    4ul      4ul     
  0.1M DTT        2ul    2ul      2ul    2ul  2ul    2ul      2ul    2ul
  0.1M DTT        2ul    2ul      2ul     
  RNaseOUT        1ul    1ul      1ul    1ul  1ul    1ul      1ul    1ul
  RNaseOUT        1ul    1ul      1ul     
  Superscript III 1ul    1ul      1ul     1ul  1ul    1ul      1ul    1ul
  Superscript III 1ul    1ul      1ul  
  Incubate all tube at 50C for 50min.
  Incubate all tube at 50C for 50min.



Revision as of 17:41, 10 June 2009

Making ds-cDNA for Hues6/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

                           A        B     C    
  RNA                     6ul     2.5ul  10ul
  10X DNase I buffer      5ul       5ul   5ul
  RNase-Free DNase I      2.5ul   2.5ul  2.5ul
  H2O                   36.5ul     40ul  32.5ul
  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

H was not DNase treated.

                 A       B       C      
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.

[edit] 2nd strand synthesis

Cleanup the first-strand reactions with G-25 columns.

To the ~40ul reactions, add    
10ul NEBuffer 2  10ul
10mM dNTP         5ul
DNA Pol I(10U/ul) 2ul
RNaseH            2ul
H2O              adjust to 100ul
Incubate at 16C for 2h, purified with Qiaquick columns.
Yield (use RNA's absorbance):
A: Hues6-ES: 50ng/ul x 30ul
B: Hues6-EB: 55ng/ul x 30ul
C: Hybrid1-ES: 30ng/ul x 30ul
D: Hybrid1-EB: 63ng/ul x 30ul
E: Hybrid2-ES: 58ng/ul x 30ul
F: Hybrid2-EB: 53ng/ul x 30ul
G: BJ:         44ng/ul x 30ul
H: Hybrid1-ES: 56ng/ul x 30ul