Kun:LabNotes/ASE/2008-1-2: Difference between revisions

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94C 3min->60C 40h-> add 1.5ul/1.0ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 1h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C hold ->  37C 1min -> add 2ul of exo mix (Exo I, Exo III, T7 exo)->37c 2 hour -> 94C 5min -> 4C hold.
94C 3min->60C 40h-> add 1.5ul/1.0ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 1h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C hold ->  37C 1min -> add 2ul of exo mix (Exo I, Exo III, T7 exo)->37c 2 hour -> 94C 5min -> 4C hold.
===Exp.2 Probe prep. for the CES22k set on two new libraries===
PCR (with iTaq)
                              x1        x16x2
        template(100nM)    0.1ul    2x1.6ul   
        10x ABI buffer      10ul      32ul
        25mM MgCl2            6ul      192ul
        10mM dNTP            2ul        64ul
        100uM pAP1V6U      0.3ul      9.6ul
        100uM phosAP2V6    0.3ul      9.6ul
        50X SYBG I          0.8ul      25.6ul
        iTaq                  1ul        32ul
        H2O                  80ul      2560ul
94C 3min -> 16 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold
Purify each amplicons with Bioneer columns.
Lambda exo digestion: to 100ul DNA, add 15ul 10X lambda exo buffer, 5ul lambda exo, 30ul H2O, 37C 4h, 75C 15min, Purify each amplicons with 2 Bioneer columns, eluted in 100 elution buffer.  Cleanup with G25 column.
Dpn II & USER digestion: Add 10ul 10X Dpn II buffer, 10ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 5ul Dpn II, 5ul USER, 37C 8h, 75C 10min.

Revision as of 01:21, 3 January 2008

Exp.1 Capturing cSNPs with the eSNP84k probe set

Templates:

  1. NA20431, 347ng/ul, one reaction with the equal molar pool of all five probe sets, 800ng/reaction.
  2. GM20431 ds-cDNA, 122ng/ul, Lib #1: 1ng; Lib #2: 20ng; Lib #3 50ng; Lib #4: 100ng; Lib #5: 400ng
  3. GC1EP, 114ng/ul, Lib #1: 10ng; Lib #2: 20ng; Lib #3 50ng; Lib #4: 100ng; Lib #5: 400ng
gDNA Hues ds-cDNA Hues(no mouse) ds-cDNA
Template 2.3 1(10ng) 1(20ng) 1(50ng) 0.85 3.4 1(10ng) 1(20ng) 1(50ng) 0.88 3.5
10x buffer 1.5 1 1 1 1 1 1 1 1 1 1
Lib #1 (384nM) 2 2 2
Lib #2 (580nM) 1.5 1.5 1.5
Lib #3 (331nM) 2.6 2.6 2.6
Lib #4 (500nM) 2.4 2.4 2.4
Lib #5 (460nM) 1.8 1.8 1.8
H2O 0.9 6 6.5 5.4 5.75 3.8 6 6.5 5.4 5.72 3.7

94C 3min->60C 40h-> add 1.5ul/1.0ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 1h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C hold -> 37C 1min -> add 2ul of exo mix (Exo I, Exo III, T7 exo)->37c 2 hour -> 94C 5min -> 4C hold.

Exp.2 Probe prep. for the CES22k set on two new libraries

PCR (with iTaq)

                             x1        x16x2 
        template(100nM)     0.1ul     2x1.6ul    
        10x ABI buffer       10ul       32ul
        25mM MgCl2            6ul       192ul
        10mM dNTP             2ul        64ul
        100uM pAP1V6U       0.3ul       9.6ul
        100uM phosAP2V6     0.3ul       9.6ul
        50X SYBG I          0.8ul      25.6ul
        iTaq                  1ul        32ul
        H2O                  80ul      2560ul

94C 3min -> 16 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold

Purify each amplicons with Bioneer columns.

Lambda exo digestion: to 100ul DNA, add 15ul 10X lambda exo buffer, 5ul lambda exo, 30ul H2O, 37C 4h, 75C 15min, Purify each amplicons with 2 Bioneer columns, eluted in 100 elution buffer. Cleanup with G25 column.

Dpn II & USER digestion: Add 10ul 10X Dpn II buffer, 10ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 5ul Dpn II, 5ul USER, 37C 8h, 75C 10min.