Kun:LabNotes/ExonomeSeq/2009-3-3: Difference between revisions
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100uM AmpR6.2Sol 0.4ul | 100uM AmpR6.2Sol 0.4ul | ||
H2O 49ul | H2O 49ul | ||
98C 30sec -> (98C 10sec -> 56C 30sec -> 72C 15sec) x | 98C 30sec -> (98C 10sec -> 56C 30sec -> 72C 15sec) x 5 -> 72C 3min -> 4C hold. | ||
Purified with Qiaquick columns, eluted with 30ul EB. | Purified with Qiaquick columns, eluted with 30ul EB. | ||
Yield: | Yield: | ||
PGP1-shaked-LONG: | PGP1-shaked-LONG: 33ng/ul x 30ul | ||
PGP1-CTRL-LONG: | PGP1-CTRL-LONG: 34ng/ul x 30ul |
Revision as of 23:22, 5 March 2009
Test whether shaking can improve the circularization
Padlock capture
- Probe sets:
- Jan09-1: 46,417 probes, 334nM
- Jan09-2: 53,493 probes, 294nM
- Jan09-3: 53,828 probes, 186nM
- Template DNA: PGP1, sheared with Bioruptor, 50ng/ul; 200ug per reaction.
- Probe:target ratio: 200:1
Shaking Control Template(50ng/ul) 4ul 4ul 10X AmpLigase buffer 2ul 2ul Jan09-1 (334nM) 3.1ul 3.1ul Jan09-2 (294nM) 4.0ul 4.0ul Jan09-3(unknown) 4.0ul 4.0ul H2O 2.0ul 2.0ul 95C 2min -> -0.02C/sec to 60C -> 60C 18h -> add 1ul SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) -> 60C 24h (move one tube to ThermoMixer @60C, 500rpm) -> 94C 1min -> 37c 1min -> add 2ul Exo I&III -> 37C 2h -> 90C 5min -> 4C hold
PCR
PCR x 2 x 2 Template: 10ul 2x iProof master mix: 50ul 100uM AmpF6.2 0.4ul 100uM AmpR6.2 0.4ul 50X SYBG I: 0.4ul H2O 39ul 98C 30sec -> (98C 10sec -> 53C 30sec -> 72C 15sec) x 16 -> 72C 3min -> 4C hold. Purified with Qiaquick columns, eluted with 30ul EB. Yield: PGP1-shaked: 28ng/ul x 30ul PGP1-CTRL: 24ng/ul x 30ul
2nd round PCR with Solexa adaptor
PCR x 2 Template: 1ul 2x iProof master mix: 50ul 100uM AmpF6.2Sol 0.4ul 100uM AmpR6.2Sol 0.4ul H2O 49ul 98C 30sec -> (98C 10sec -> 56C 30sec -> 72C 15sec) x 5 -> 72C 3min -> 4C hold. Purified with Qiaquick columns, eluted with 30ul EB. Yield: PGP1-shaked-LONG: 33ng/ul x 30ul PGP1-CTRL-LONG: 34ng/ul x 30ul