AlanFung:LabNotes/DNA/2009-8-18

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Revision as of 22:44, 18 August 2009 by >Alan6017518 (→‎quantification with qPCR)
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Digestion with MmeI

PGP4 PGP6 PGP10 A B
Sample 20 20 20 10 10
10X NEBuffer 4 4 4 4 3 3
1mM SAM (fresh) 8 8 8 6 6
2U/ul MmeI 1 1 1 1 1
ddH20 7 7 7 10 10
Total 40 40 40 30 30
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h. 
  • Qiaquick column purification. Elute in 12ul EB.

quantification with qPCR

I dilute the PhiX lib(10nM) to 1nM, 0.5nM, 0.25nM, 0.05nM.
I dilute the Parkinson's lib with 1:10 ratio. 
Syb_FP5: ATGATACGGCGACCACCGAG
Syb_RP7: CAAGCAGAAGACGGCATACGAG
1nM PhiX 1:10 PGP4 1:10 PGP10 1:10 50192B
1nM PhiX 1:10 PGP4 1:10 PGP10 1:10 50192B
0.1nM PhiX 1:50 PGP4 1:50 PGP10 1:50 50192B
0.1nM PhiX 1:50 PGP4 1:50 PGP10 1:50 50192B
0.025nM PhiX 1:10 PGP6 1:10 50192A
0.025nM PhiX 1:10 PGP6 1:10 50192A
0.005nM PhiX 1:50 PGP6 1:50 50192A
0.005nM PhiX 1:50 PGP6 1:50 50192A
X29
Template 1ul
2X iProof Mix 25 725
SYB_RP7 (100uM) 0.2 5.8
SYB_FP5 (100uM) 0.2 5.8
H2O 24
50X SYBR I 0.2 5.8

98C 30sec -> 10 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) ->72C 3min -> 15C hold.