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

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* Follow the protocol: [http://arep.med.harvard.edu/pdf/Porreca07.pdf] and standard protocol using to make DMR330k probe set<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 concentration 20 nM (in case it is lyophilized)
* After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized)
* perform expansion PCR to amplify the probe as the template<br>
* perform expansion PCR to amplify the probes as the template<br>


== Expansion PCR ==
== Expansion PCR ==

Revision as of 14:24, 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 concentration 20 nM (in case it is lyophilized)
  • perform expansion PCR to amplify the probes 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

  • Primer info.
    • eMIP_CA1_F: TGCCTAGGACCGGATCAACT (TGCCT overhang), Tm of the short one = 53.06C, the long one = 63.28C
    • eMIP_CA1_R: GAGCTTCGGTTCACGCAATG (GAGCT overhang), Tm of the short one = 54.41C, the long one = 62.95C
  • Note:
    • The first three cycles, Tm of each primer was calculated without 5 nt overhang, then the Tm was increase based on the Tm of the full length primer
    • 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