Kun:LabNotes/ASE/2007-11-20

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Exp. 1 Perform MDA on the gDNA from BJ Fibroblast[edit]

       Template DNA(6ng/ul)  20ul
       10X RepliPhi buffer  2.5ul
       1mM N6               2.5ul
       95C 3min -> chill on ice -> add the reaction mix:    
       10X RepliPhi buffer 8.5ul
       25mM dNTP             4ul
       1mM N6              2.5ul
       H2O                  49ul
       100mM DTT             5ul
       RepliPhi phi-29       5ul
       30C 3h -> 85C 10min
       Purify with Microcon YM-100 => 576ng/ul

Exp. 2 Perform CES22k eSNP capture on PGP and hybrid samples[edit]

Template 10x buffer CES22k probes (277nM) H2O
1 GM20431 gDNA(347ng/ul) 2.3ul 1ul 5.4ul 0.3ul
2 GM20431 cDNA (121ng/ul) 0.4ul 1ul 5.4ul 2.2ul
3 GC1F cDNA(48ng/ul) 1ul 1ul 5.4ul 1.6ul
4 GC1EP cDNA(52ng/ul) 1ul 1ul 5.4ul 1.6ul
5 Hues6 gDNA(113ng/ul) 2.6ul 1ul 5.4ul 0ul
6 Hues6 cDNA(155ng/ul) 0.3ul 1ul 5.4ul 2.3ul
7 H1 gDNA(222ng/ul) 2.6ul 1ul 5.4ul 0ul
8 H1 cDNA(35ng/ul) 1.4ul 1ul 5.4ul 1.2ul
9 H2 gDNA(200ng/ul) 2.6ul 1ul 5.4ul 0ul
10 H2 cDNA(48ng/ul) 1ul 1ul 5.4ul 1.6ul
11 BJ MDA DNA(576ng/ul) 2.6ul 1ul 5.4ul 0ul
12 BJ cDNA(18ng/ul) 2.6ul 1ul 5.4ul 0ul

94C 5min->60C 18h-> add 0.5ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 4h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C 4h -> 37C 1min -> add 1ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.

[11-22-2007] PCR

                              one reaction       12 reactions
          Template:              5ul                  each
          10x Buffer:          9.5ul               123.5ul
          25mM MgCl2             6ul                  78ul
          25mM dNTP            1.2ul                15.6ul
          100uM AmpSolV6F2     0.4ul                 5.2ul
          100uM AmpSolV6R2     0.4ul                 5.2ul
          Platinium Taq          1ul                  13ul
          50X SYBG I           0.8ul                10.4ul
          H2O                 76.7ul                 997ul

94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.

   Added 3 more cycles for #3, 4, 6, 8, 10, 11, 12. 
   File:2007-11-24-CES22k-capture-PCR.png
   Since the amplification curve for #1 came up much earlier than others, I repeat the PCR on 3ul #1 with 10 cycles at the second stage of amplification.