Noi:LabNotes/2010-10-21: Difference between revisions
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Line 12: | Line 12: | ||
100ul x 96 | 100ul x 96 | ||
95C 30sec | 95C 30sec | ||
(95C 3sec -> 55C 30sec-> 60C 20sec) x 15 cycles | |||
60C 2min | |||
15C hold. | |||
Ethanol precipitation; re-purified with twelve Qiaquick columns, each eluted with 40ul EB (total vol: 12 x 40 = 480ul). | 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) | Amplified amplicon conc. (ng/ul) 194.2 192.7 | ||
O.D.260/280 | O.D.260/280 1.93 1.92 | ||
Yield: conc. x total vol. | Yield: conc. x total vol. 194.2ng/ulx480ul= 93.2ug 192.7ng/ulx480ul = 92.5ug | ||
===Lambda exo digestion=== | ===Lambda exo digestion=== | ||
'''DMR220k_S4''' '''DMR220k_S5''' | |||
Qiaquick amplified amplicon (approx. 20ug) 16 15 | Qiaquick amplified amplicon (approx. 20ug) 16 15 | ||
10x Lambda Exo buffer | 10x Lambda Exo buffer |
Revision as of 07:40, 23 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 15 cycles 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) 194.2 192.7 O.D.260/280 1.93 1.92 Yield: conc. x total vol. 194.2ng/ulx480ul= 93.2ug 192.7ng/ulx480ul = 92.5ug
Lambda exo digestion
DMR220k_S4 DMR220k_S5 Qiaquick amplified amplicon (approx. 20ug) 16 15 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.