AlanFung:LabNotes/DNA/2009-8-18

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Revision as of 16:33, 20 August 2009 by >Alan6017518 (→‎Digestion with MmeI)
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Digestion with MmeI[edit]

PGP4 PGP6 PGP10 5012A 5012B
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 30ul EB.

quantification with qPCR[edit]

  • Dilute the PhiX lib(10nM) to 1nM, 0.1nM, 0.025nM, 0.005nM
  • Dilute the samples to 1:10 and 1:50 ratio
  • 1:10-Add 1ul of sample to 9ul H20
  • 1:50-Add 1ul of 1:10 sample to 4ul H20
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 24 696
50X SYBR I 0.2 5.8
Toal 1438.4

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

  • stopped when curve reached plateau
' nM sample ul Tris-CL ul HP3 total
PGP4 1.71 10 6.25 0.86 17.1
PGP6 1.21 10 1.5 0.61 12.1
PGP10 1.56 10 4.82 0.78 15.6
50192A 2.44 10 13.18 1.22 24.4
50192B 1.4 10 3.3 0.7 14
PGP6 1.21 20 2.99 1.21 24.2