Noi/NOTES/2014-3-26: Difference between revisions
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>Noi (Created page with "= MONOD_V3_12k probe preparation = '''2013-03-26''' : - Received oligos from CustomArray : '''Oligo information''' :: - Design: BSPP_Design-5-17-2013 --> I would name BSPP_Blu...") |
>Noi mNo edit summary |
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95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold | 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) | : - Purified 1st round amplicon in 2 QIAquick column and elute with EB buffer 50ul each (Final volume left ~95ul) | ||
: - N.D. = 7.35ng/ul | : - N.D. = 7.35ng/ul | ||
: - Dilute 1st round amplicon to 20nM | :: 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 == | == Production PCR == | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | Program | ||
Revision as of 15:56, 30 March 2014
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