Kun:LabNotes/ASE/2007-12-5: Difference between revisions
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(New page: ==Exp. 1 Perform PCR on the capturing reactions started on 12/03/2007== one reaction 3 reactions Template: 5ul ...) |
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94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold. | 94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold. | ||
==Exp.2 Perform real-time PCR to quantify the efficiency of the capturing reactions started at 12-03-07 and 11-21-07== | |||
Per Billy's suggestion, I'm using a diluted Solexa library with known concentration as the reference to quantify the circularization efficiency. | |||
<b>Reference:</b> H1 gDNA Solexa library (11-27-07), 20.6nM, series diluted to 100pM, 10pM, 1pM, 100fM. <br> | |||
<b>Template:</b> | |||
#12-03-07: H1 cDNA, H2 cDNA, Hues6 cDNA, 1ul each | |||
#11-26-07: BJ cDNA 1ul | |||
#11-21-07: H1 cDNA, H2 cDNA, Hues6 cDNA, BJ cDNA, 1ul each | |||
Two 50ul reactions for each template. | |||
one reaction 24 reactions | |||
Template: 1ul each | |||
10x Buffer: 5ul 120ul | |||
25mM MgCl2 3ul 72ul | |||
25mM dNTP 0.6ul 14.4ul | |||
100uM AmpV6F 0.2ul 4.8ul | |||
100uM AmpV6R 0.2ul 4.8ul | |||
iTaq 0.5ul 12ul | |||
50X SYBG I 0.4ul 9.6ul | |||
H2O 39ul 936ul | |||
94C 3min -> 30 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 72C 3min -> 15C hold. |
Revision as of 19:02, 5 December 2007
Exp. 1 Perform PCR on the capturing reactions started on 12/03/2007
one reaction 3 reactions Template: 5ul each 10x Buffer: 9.5ul 28.5ul 25mM MgCl2 6ul 18ul 25mM dNTP 1.2ul 3.6ul 100uM AmpSolV6F2 0.4ul 1.2ul 100uM AmpSolV6R2 0.4ul 1.2ul Platinium Taq 1ul 3ul 50X SYBG I 0.8ul 2.4ul H2O 76.7ul 231ul
94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.
Exp.2 Perform real-time PCR to quantify the efficiency of the capturing reactions started at 12-03-07 and 11-21-07
Per Billy's suggestion, I'm using a diluted Solexa library with known concentration as the reference to quantify the circularization efficiency.
Reference: H1 gDNA Solexa library (11-27-07), 20.6nM, series diluted to 100pM, 10pM, 1pM, 100fM.
Template:
- 12-03-07: H1 cDNA, H2 cDNA, Hues6 cDNA, 1ul each
- 11-26-07: BJ cDNA 1ul
- 11-21-07: H1 cDNA, H2 cDNA, Hues6 cDNA, BJ cDNA, 1ul each
Two 50ul reactions for each template.
one reaction 24 reactions Template: 1ul each 10x Buffer: 5ul 120ul 25mM MgCl2 3ul 72ul 25mM dNTP 0.6ul 14.4ul 100uM AmpV6F 0.2ul 4.8ul 100uM AmpV6R 0.2ul 4.8ul iTaq 0.5ul 12ul 50X SYBG I 0.4ul 9.6ul H2O 39ul 936ul
94C 3min -> 30 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 72C 3min -> 15C hold.