User:DrJones1935/16 July 2012

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*See [[MAGE_in_Naturally_Competent_Organisms#cT5_.28normal.29|Project Page]] for more info
 
====II. Resuspend Lyophilized Primers for Cross-over PCR====
====II. Resuspend Lyophilized Primers for Cross-over PCR====

Latest revision as of 01:55, 4 October 2012

I. Ordered Cross-over PCR Primers from Keck

  • Primers were ordered with the name/sequence in the following table:
Name Tm (Tm overhang/Tm anneal) Length (bp) Sequence (overCUTSITEanneal)
cT5_pAf 66.64 (25.23/58.79) 30 (10/20) 5'-tgacTCTAGAtttgctttgtgagcggataa-3' (XbaI)
cT5_pAr 69.08 (30.28/55.61) 25 (10/15) 5'-gtaaCTGCAGgagagcgttcaccga-3' (PstI)

II. Resuspend Lyophilized Primers for Cross-over PCR

  • Spin down tubes
  • Add the volume specified in the table of TE Buffer to the primer tubes
Name Volume (uL)
p1f 208.35
p1r 217.78
p2f 199.75
p2r 199.6
p3f 199.75
p3r 190.55
p4f 248.1
p4r 149.85
p5f 127.75
p5r 199.65
  • Vortex all tubes briefly
  • Spin all tubes down
  • Incubate on bench for 10 minutes
  • Store at -20 oC
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