Jie:LabNotes/ASE/2009-6-10

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