Noi/NOTES/2014-3-26
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MONOD_V3_12k probe preparation
2013-03-26
- - Received oligos from CustomArray
- Oligo information
- - Design: BSPP_Design-5-17-2013 --> I would name BSPP_Blueprint for future experiment
- - Length 146 mer (MW = 146 * 330Da/bp = 48,180 Da or g/mole
- - Cocn. 30.35ng/ul = 629 nM (30.35ng/ul/ 48,180 g/mole)
- - Volume 80ul in TE buffer (The actual volume is smaller than 80ul
Expansion PCR
Components | Volume (ul) | Final conc. | Volume for 1.5 rxn mix |
Seed oligo (629nM) | 15.90 | 100nM | 23.85 |
AP1V51U (100uM) | 0.40 | 400nM | 0.60 |
AP2V6 (100uM) | 0.40 | 400nM | 0.60 |
2x KAPA SYBG fast MM | 50.00 | 1x | 75.00 |
H2O | 33.30 | 49.95 | |
Total | 100.00 | 150.00 |
- Aliquot 75ul to 2 of WW tubes
Program
95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold * I stopped reaction at 13 cycles
- - Purified 1st round amplicon in 2 QIAquick column and elute with EB buffer 50ul each (Final volume left ~95ul)
- - N.D. = 7.35ng/ul
- MW of dsDNA 146mer = 146*660 = 96360g/mole
- 7.35ng/ul = 14.7ng/ul/96360g/mole/10^6 = 152.55nM
- - Dilute 1st round amplicon to 20nM by mixing 90ul of 14.7ng/ul 1st round amplicons with 596.48ul TE buffer
- I 20nM 1st round amplicons as template instead of 10nM since oligo pools from CustomArray has very high background even after expansion PCR. I use the same volume for probe production.
Production PCR
Components | 1 rxn | 50x rxn mix |
1st round amplicon (20nM) | 0.20 | 10.00 |
AP1V61U (100uM) | 0.40 | 20.00 |
AP2V6 (100uM) | 0.40 | 20.00 |
2x KAPA SYBG fast MM | 50.00 | 2500.00 |
H2O | 49.00 | 2450.00 |
Total | 100.00 | 5000.00 |
- Aliquot 100ul to each well in half of 96-well plate
Program 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold
* I stopped at 14 cycles