Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/12

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Overview

Taking the following steps today:

  • PCR of EM7:ZeoR to add BB cut sites
  • Digestion/Ligation of EM7:ZeoR part into v0120 vector
  • Transformation into DH5α-Turbo

EM7:ZeoR PCR

Primers:

primer name sequence Tm (anneal)
EM7_BB_fwdCCTTTCTAGATGTTGACAATTAATCATCGGCATAGTATATCGGC60.6
ZeoR_BB_revAAGGCTGCAGCGGCCGCTACTAGTTCAGTCCTGCTCCTCGGCC60.1

Reaction setup:

Product Primer (For) Primer (Rev) Template
EM7:ZeoR_BBEM7_BB_fwdZeoR_BB_revMV8

Prepare master mix:

Reagent Stock Volume (µL)
GC Buffer5x20
dNTP mix10 mM2
DMSO-3
Primer (for)100 µM1.0
Primer (rev)100 µM1.0
Template100 ng/µL4.0
Phusion20 U/µL1
H2O-68
100

PCR extension times:

Part Size (bp) Extension time (sec)
EM7:ZeoR_BB47630

DNA Quantification

Concentration is measured at 245 ng/µL with 260/280 around 1.9.


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