Kun:LabNotes/ASE/2008-1-11

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Exp. 1 RNA clean-up & RT on GC1F, Hues30[edit]

Received GC1F from Jay Lee today. The shipping was delayed but there was still dry ice in the box, so RNA should be fine.

RNA clean-up[edit]

Clean up 20ul GC1F total RNA (0.5ug/ul) and 10ul Hues30 total RNA (0.8ug/ul) with RNeasy Plus columns. Yield:

  • GC1F: 200ng/ul x 30ul
  • Hues30: 60ng/ul x 30ul

Also did DNase I digestion on GC1F total RNA. 8ul 0.5ug/ul total RNA + 1ul 10X DNase I buffer + 1ul DNase I (RNase free), 5U/ul 37C 30min -> 80C 20min.

Reverse transcription[edit]

        GC1F(0.5ug/ul)   GC1F(0.2ug/ul)  GC1F(DNase)  Hues30(60ng/ul)
 RNA          4ul             25ul           5ul         25ul
 10mM dNTP    1ul              3ul           1ul                                       3ul
 50uM d(T)    1ul              3ul           1ul          3ul
 H2O          4ul              0ul           3ul          0ul
 65C 5min -> place on ice for > 1min
 Add
 10X RT buffer 2ul             6ul           2ul          6ul
 25mM MgCl2    4ul            12ul           4ul         12ul
 0.1M DTT      2ul             6ul           2ul          6ul
 RNase OUT     1ul             2ul           1ul          2ul
 SS III        1ul             3ul           1ul          3ul
 37C 10min -> 50C 50min -> 85C 5min

Second strand synthesis[edit]

Clean up the first strand reactions with G-25 columns.

 to the 20/60ul reactions, add
 H2O (DEPC)         11ul/22ul
 10X NEBuffer 2     4ul/10ul 
 10mM dNTP          2ul/3ul
 DNA Pol I          2ul/4ul
 RNase H            1ul/1ul
 Mix well, incubate at 16C for 2h, purified with MinElute columns.
 Yield:
           57ng/ul          167ng/ul          44ng/ul          44ng/ul

Exp.2 PCR on the eSNP84k capturing reactions started on 01/08/08[edit]

                          x 1         x 14
     template:           10ul         each
     JumpStart buffer:    9ul        126ul
     10mM dNTP:           2ul         28ul
     100uM AmpF2Sol:    0.4ul        5.6ul
     100uM AmpRSol:     0.4ul        5.6ul
     50X SYBG I:        0.8ul       11.2ul
     JumpStart:           2ul         28ul
     H2O:                75ul       1050ul

94C 3min -> 8 cycles of (94C 45sec -> 56C 2min -> 72C 1min) -> 10 cycles of (94C 45 sec -> 72C 1min) -> 72C 3min -> 15C hold.