Kun:LabNotes/ASE/2007-11-20: Difference between revisions

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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 3ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.
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 3ul 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 -> 58C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.

Revision as of 18:24, 22 November 2007

Exp. 1 Perform MDA on the gDNA from BJ Fibroblast

       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

Template 10x buffer CES22k probes (277nM) H2O
GM20431 gDNA(347ng/ul) 2.3ul 1ul 5.4ul 0.3ul
GM20431 cDNA (121ng/ul) 0.4ul 1ul 5.4ul 2.2ul
GC1F cDNA(48ng/ul) 1ul 1ul 5.4ul 1.6ul
GC1EP cDNA(52ng/ul) 1ul 1ul 5.4ul 1.6ul
Hues6 gDNA(113ng/ul) 2.6ul 1ul 5.4ul 0ul
Hues6 cDNA(155ng/ul) 0.3ul 1ul 5.4ul 2.3ul
H1 gDNA(222ng/ul) 2.6ul 1ul 5.4ul 0ul
H1 cDNA(35ng/ul) 1.4ul 1ul 5.4ul 1.2ul
H2 gDNA(200ng/ul) 2.6ul 1ul 5.4ul 0ul
H2 cDNA(48ng/ul) 1ul 1ul 5.4ul 1.6ul
BJ MDA DNA(576ng/ul) 2.6ul 1ul 5.4ul 0ul
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 3ul 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 -> 58C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.