Noi/NOTES/2011-10-6: Difference between revisions
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>Noi (Created page with '== Plan of probe (from LC Bioscience) production == * Follow the protocol: [http://arep.med.harvard.edu/pdf/Porreca07.pdf]<br> * After receiving the probes, resuspend with H2O to…') |
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== Plan of probe (from LC Bioscience) production == | == Plan of probe (from LC Bioscience) production == | ||
* Follow the protocol: [http://arep.med.harvard.edu/pdf/Porreca07.pdf]<br> | * Follow the protocol: [http://arep.med.harvard.edu/pdf/Porreca07.pdf] and standard protocol using to make DMR330k probe set<br> | ||
* After receiving the probes, resuspend with H2O to the concemtration 20 nM | * After receiving the probes, resuspend with H2O to the concemtration 20 nM | ||
* perform expansion PCR to amplify the probe as the template<br> | * perform expansion PCR to amplify the probe as the template<br> | ||
== Expansion PCR == | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
Line 8: | Line 9: | ||
| align="center" style="background:#f0f0f0;"|'''Final conc.''' | | align="center" style="background:#f0f0f0;"|'''Final conc.''' | ||
|- | |- | ||
| | | 20nM LC Sciences Oligoes||2.00||0.2nM | ||
|- | |- | ||
| eMIP_CA1_F (100uM)||0.80||400nM | | eMIP_CA1_F (100uM)||0.80||400nM | ||
Line 14: | Line 15: | ||
| eMIP_CA1_F (100uM)||0.80||400nM | | eMIP_CA1_F (100uM)||0.80||400nM | ||
|- | |- | ||
| 2x | | 2x Kapa SYBG MM||100.00||1x | ||
|- | |- | ||
| H2O|| | | H2O||96.40|| | ||
|- | |- | ||
| Total||200.00|| | | Total||200.00|| | ||
|} | |} | ||
'''Program''' | '''Program'''<br> | ||
95C 30sec -> (95C 3sec -> 48C 30sec-> 60C 20sec) x 3 -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 13 -> 60C 2min -> 15C hold | |||
* Purified with Qaiquick column | * Note: | ||
* Measure DNA conc. with Nanodrop and adjust conc. to | ** The first three cycles, Tm of each primer was calculated without 5 nt overhang, the the Tm was increase based on the Tm of the whole primer length | ||
** The number of cycles will be monitored | |||
* Purified with Qaiquick column and elute with EB buffer volume 50ul each | |||
* Measure DNA conc. with Nanodrop and adjust conc. to 10nM for using as the template for the future amplification | |||
* Perform production PCR | |||
== Production PCR == | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''32x rxn mix''' | |||
|- | |||
| 1st round amplicon (10nM)||0.20||6.40 | |||
|- | |||
| eMIP_CA1_F (100uM)||0.40||12.80 | |||
|- | |||
| eMIP_CA1_F (100uM)||0.40||12.80 | |||
|- | |||
| 2x Kapa SYBG MM||50.00||1600.00 | |||
|- | |||
| H2O||49.00||1568.00 | |||
|- | |||
| Total||100.00||3200.00 | |||
|} | |||
'''Program'''<br> | |||
95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 16 -> 60C 2min -> 15C hold | |||
* The number of cycles will be monitored to prevent overamplification |
Revision as of 10:41, 7 October 2011
Plan of probe (from LC Bioscience) production
- Follow the protocol: [1] and standard protocol using to make DMR330k probe set
- After receiving the probes, resuspend with H2O to the concemtration 20 nM
- perform expansion PCR to amplify the probe as the template
Expansion PCR
Components | Volume (ul) | Final conc. |
20nM LC Sciences Oligoes | 2.00 | 0.2nM |
eMIP_CA1_F (100uM) | 0.80 | 400nM |
eMIP_CA1_F (100uM) | 0.80 | 400nM |
2x Kapa SYBG MM | 100.00 | 1x |
H2O | 96.40 | |
Total | 200.00 |
Program
95C 30sec -> (95C 3sec -> 48C 30sec-> 60C 20sec) x 3 -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 13 -> 60C 2min -> 15C hold
- Note:
- The first three cycles, Tm of each primer was calculated without 5 nt overhang, the the Tm was increase based on the Tm of the whole primer length
- The number of cycles will be monitored
- Purified with Qaiquick column and elute with EB buffer volume 50ul each
- Measure DNA conc. with Nanodrop and adjust conc. to 10nM for using as the template for the future amplification
- Perform production PCR
Production PCR
Components | 1 rxn | 32x rxn mix |
1st round amplicon (10nM) | 0.20 | 6.40 |
eMIP_CA1_F (100uM) | 0.40 | 12.80 |
eMIP_CA1_F (100uM) | 0.40 | 12.80 |
2x Kapa SYBG MM | 50.00 | 1600.00 |
H2O | 49.00 | 1568.00 |
Total | 100.00 | 3200.00 |
Program
95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 16 -> 60C 2min -> 15C hold
- The number of cycles will be monitored to prevent overamplification