Noi:LabNotes/2010-10-21: Difference between revisions
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DMR220k_S4 DMR220k_S5 | |||
Amplified amplicon conc. (ng/ul) | |||
O.D.260/280 | O.D.260/280 | ||
Yield: | Yield: conc. x total vol. | ||
===Lambda exo digestion=== | |||
DMR220k_S4 DMR220k_S5 | |||
Qiaquick amplified amplicon (approx. 20ug) | |||
10x Lambda Exo buffer | |||
Lambda exo | |||
H20 | |||
-> 37C 1h -> purified with four Qiaquick columns, each eluted with 60ul EB (total vol: 4 x 60 = 240ul). | |||
DMR220k_S4 DMR220k_S5 | |||
Single-strand(SS) amplicon conc. (ng/ul) | |||
O.D.260/280 | |||
Yield: conc. x total vol. |
Revision as of 15:21, 22 October 2010
Probe prep for the DMR220k_S4 and S5
(followed Dr. Zhang's conditions)
Production PCR
10nM 1st round amplicon 0.2ul 2x Kapa SYBG qPCR Master Mix 50ul 100uM pAP1V61U 0.4ul 100uM AP2V6 0.4ul H2O 49ul =================================== 100ul x 96
95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 16 -> 60C 2min -> 15C hold. Ethanol precipitation; re-purified with twelve Qiaquick columns, each eluted with 40ul EB (total vol: 12 x 40 = 480ul).
DMR220k_S4 DMR220k_S5 Amplified amplicon conc. (ng/ul) O.D.260/280 Yield: conc. x total vol.
Lambda exo digestion
DMR220k_S4 DMR220k_S5
Qiaquick amplified amplicon (approx. 20ug) 10x Lambda Exo buffer Lambda exo H20
-> 37C 1h -> purified with four Qiaquick columns, each eluted with 60ul EB (total vol: 4 x 60 = 240ul).
DMR220k_S4 DMR220k_S5 Single-strand(SS) amplicon conc. (ng/ul) O.D.260/280 Yield: conc. x total vol.