Kun:LabNotes/ASE/2007-11-9

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Exp.1 eSNPs capture (cont.)[edit]

Perform PCR on 5ul capturing reactions.

                          x 1        x 12
     template:            5ul         each
     ABI 10x buffer:    4.5ul         54ul
     25mM MgCl2:          3ul         36ul
     10mM dNTP:           1ul         12ul
     100uM AmpF2Sol:   0.15ul        1.8ul
     100uM AmpRSol:    0.15ul        1.8ul
     50X SYBG I:        0.4ul        4.8ul
     iTaq:              0.5ul          6ul
     H2O:              35.3ul        430ul

94C 3min -> 10 cycles of (94C 45sec -> 56C 2min -> 72C 1min) -> 20 cycles of (94C 45 sec -> 72C 1min) -> 72C 3min -> 15C hold.

 File:2007-11-09-eSNP84k-capture-PCR.png

Repeat PCR on 10ul capturing reactions using Invitrogen's Platinum Taq.

                          x 1        x 12
     template:           10ul         each
     ABI 10x buffer:      9ul        108ul
     25mM MgCl2:          6ul         72ul
     10mM dNTP:           2ul         24ul
     100uM AmpF2Sol:    0.3ul        3.6ul
     100uM AmpRSol:     0.3ul        3.6ul
     50X SYBG I:        0.6ul        9.6ul
     Platinum Taq:        1ul         12ul
     H2O:              72.4ul        869ul

94C 3min -> 10 cycles of (94C 45sec -> 56C 2min -> 72C 1min) -> 13 cycles of (94C 45 sec -> 72C 1min) -> take out the gDNA tube -> 9 cycles of (94C 45 sec -> 72C 1min)-> 72C 3min -> 15C hold.

  File:2007-11-09-eSNP84k-capture-PCR2.png

Since some tubes in this run amplified better than the others, some of the reactions might be saturated. I decided to set up another PCR on the remaining capturing reactions.

                          x 1        x 10
     template:            5ul         each
     ABI 10x buffer:    4.5ul         45ul
     25mM MgCl2:          3ul         30ul
     10mM dNTP:           1ul         10ul
     100uM AmpF2Sol:   0.15ul        1.5ul
     100uM AmpRSol:    0.15ul        1.5ul
     50X SYBG I:        0.4ul          4ul
     Platinum Taq:        1ul         10ul
     H2O:              35.3ul        353ul

94C 3min -> 10 cycles of (94C 45sec -> 56C 2min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72C 1min) -> 72C 3min -> 15C hold.

 File:2007-11-09-eSNP84k-capture-PCR3.png

Exp. 2 Quantify the second batch of eSNP84k padlock probes[edit]

    eSNP84k set 1: 5.8ng/ul = 223nM
    eSNP84k set 2: 7.3ng/ul = 281nM
    eSNP84k set 3: 8.6ng/ul = 331nM
    eSNP84k set 4: 8.0ng/ul = 308nM 
    eSNP84k set 5: 6.4ng/ul = 246nM