Noi/NOTES/2011-10-6: Difference between revisions

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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.'''
|-
|-
| Resuspended probes (100nM)||5.00||2.5nM
| 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 Phusion master mix||100.00||1x
| 2x Kapa SYBG MM||100.00||1x
|-
|-
| H2O||93.40||
| H2O||96.40||
|-
|-
| Total||200.00||
| Total||200.00||
|}<br>
|}
'''Program'''
'''Program'''<br>
98C 30s -> (98C 10s -> 55C 20s -> 72C 20s)x16 -> 72C 5 min -> 15C hold<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 (2 columns) and elute with EB buffer volume 50ul each -> total volume 100ul.
* Note:
* Measure DNA conc. with Nanodrop and adjust conc. to 100nM
** 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