Matthew Cai:LabNotes/Probe Production and Capture: Difference between revisions

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| Total||200.00||
| Total||200.00||
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'''Program'''
'''Program'''<br>
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x '''5''' -> (95C 5sec -> 60C 30sec-> 72C 30sec) x '''10''' -> 72C 2min -> 15C hold
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x '''5''' -> (95C 5sec -> 60C 30sec-> 72C 30sec) x '''10''' -> 72C 2min -> 15C hold
* Primer info.  
* Primer info.  

Revision as of 01:00, 27 February 2013

Experimental Plan

Oligo Resuspension

  • After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized)
  • Perform expansion PCR to amplify the oligoes as the template

Oligo Expansion

Components Volume (ul) Final conc.
20nM LC Sciences Oligoes 10.00 1nM
eMIP_CA1_F (100uM) 0.80 400nM
eMIP_CA1_R (100uM) 0.80 400nM
2x Kapa SYBG MM 100.00 1x
H2O 88.40
Total 200.00

Program
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 10 -> 72C 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 five cycles, Tm of each primer was calculated without 5 nt overhang, then the Tm was increased based on the Tm of the full length primer
    • The number of cycles will be monitored
  • Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul
  • Measure DNA conc. with Nanodrop: 18ng/ul or 295.5nM
  • Dilute 1st round amplicon to 10nM volume 1000ul (mix 33.89ul of 295.5nM 1st round amplicon with 966.11ul H2O)--> for using as the template for the future amplification

Production PCR