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