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