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

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==Making ds-cDNA for Hues6/Hybrids/BJ==
==Making ds-cDNA for Hybrids/BJ==


     * Total RNA Samples:
     * Total RNA Samples:
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Reverse transcription
Reverse transcription
H was not DNase treated.


                   A      B      C       
                   A      B      C       
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  Incubate all tube at 50C for 50min.
  Incubate all tube at 50C for 50min.


[edit] 2nd strand synthesis
2nd strand synthesis


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


  To the ~40ul reactions, add     
  To the 30ul reactions, add     
  10ul NEBuffer 2 10ul
  10ul NEBuffer 2   5ul
  10mM dNTP        5ul
  10mM dNTP        2.5ul
  DNA Pol I(10U/ul) 2ul
  DNA Pol I(10U/ul) 1ul
  RNaseH            2ul
  RNaseH            1ul
  H2O              adjust to 100ul
  H2O              10.5ul
  Incubate at 16C for 2h, purified with Qiaquick columns.
  Incubate at 16C for 2h, purified with Qiaquick columns.
  Yield (use RNA's absorbance):
 
  A: Hues6-ES: 50ng/ul x 30ul
  Yield (use DNA's absorbance):
B: Hues6-EB: 55ng/ul x 30ul
  A: Hybrid1-ES(750ng/ul): 22.9ng/ul x 30ul
  C: Hybrid1-ES: 30ng/ul x 30ul
  B: Hybrid1-ES(1.9ug/ul): 80.2ng/ul x 30ul
D: Hybrid1-EB: 63ng/ul x 30ul
  C: BJ(520ng/ul):        94.4ng/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

Revision as of 17:58, 11 June 2009

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

                           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

                 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.

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.
Yield (use DNA's absorbance):
A: Hybrid1-ES(750ng/ul): 22.9ng/ul x 30ul
B: Hybrid1-ES(1.9ug/ul): 80.2ng/ul x 30ul
C: BJ(520ng/ul):         94.4ng/ul x 30ul