Jie:LabNotes/ASE/2009-6-10

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Making ds-cDNA for Hybrids/BJ[edit]

   * 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):                     RNA
A: Hybrid1-ES(750ng/ul): 22.9ng/ul x 30ul         18.1ng/ul
B: Hybrid1-ES(1.9ug/ul): 80.2ng/ul x 30ul         63.4ng/ul
C: BJ(520ng/ul):         94.4ng/ul x 30ul         72.3ng/ul

capture with CES27k9bp[edit]

                B    C    
 Sample       8ul  8ul   
 10x buffer   2ul  2ul    
 CES27k9bp    5ul  5ul  
 H2O          5ul  5ul 

SLN mixture:

                                       ddH2O  Ligase Buffer   Ligase   Amplitaq    dNTP  
 2U/ul Stoffel;0.5U/ulLigase;0.5uM dNTP  0.1ul  0.1ul       0.1ul       0.2ul    (1uM)0.5ul    
                                x20      2ul      2ul         2ul         4ul     10ul
  95C 2min -> 60C 24h -> add 2ul SLN mix (dNTP 0.5uM, Stoffel 2U/ul, AmpLigase 1U/ul) 
    -> 60C 18h -> 95C 5min -> 37c 1min -> add 2ul Exo I&III
    -> 37C 1h -> 94C 5min -> 4C hold

PCR[edit]

 Template                  10ul       x4
 2X iProof Mastermix     50ul      200ul
 AmpF6.2SoL (10uM)        4ul       16ul
 AmpR6.2SoL (10uM)        4ul       16ul   
 50X SYBG I             0.4ul      1.6ul
 H2O                   31.6ul    126.4ul

I did 20 cycles for BJ and 21 cycles for Hybrid_1.

File:20090615 CES27k9bp Hybrid1 BJ.jpg20090615_CES27k9bp_Hybrid1_BJ

Qiaquick purification and gel quantification:

Hybrid_1: 8.6ng/ul x 30ul BJ: 14.3ng/ul x 30ul

I send the two samples to Zhen Ye as follows:

 Hybrid_1: 4.3ng/ul (37nM) x 5ul
 BJ: 7.2ng/ul (62nM) x 5ul