Matt:LabNotes/2014-9-18

From ZhangLabWiki
Revision as of 21:09, 19 September 2014 by >Mzcai (Created page with "==Agi26k0gap Probe Production== ===Production PCR=== *Make Master Mix for 51 well (7 strips) *0 gap first round amplicon -> V6 primers {| {{table}} | align="center" style="b...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

Agi26k0gap Probe Production

Production PCR

  • Make Master Mix for 51 well (7 strips)
  • 0 gap first round amplicon -> V6 primers
Components 1x rxn (ul) 50x rxn (ul)
First round amplicon (10nM) 0.2 10
2x Kapa SYBR MM 50 2500
100uM pAP1V6U 0.4 20
100uM AP2V6 0.4 20
H2O 49 2450
Total 100 5000

98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold

650px

EtOH Precipitation

  • 6 5-ml tubes (with 8.5 wells of PCR product each) for 0gap
    • 850ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 85ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight