Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/16

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Overview

To do:

  • Digest EM7:ZeoR_BB with XbaI & PstI
  • Gel purify the digested insert (~450bp)
  • Ligation with X&P digested v0120 backbone
  • Transformation into DH5α-Turbo competent E. coli

Digestion

Reagent Volume (µL)
EM7:ZeoR_BB (245 ng/µL) 15
FD Buf 10x GRN 3
XbaI 1
PstI 1
H2O 10
Total 30

Incubate at 37 °C for 10 minutes. Run the reaction on a gel at 100 V for 45 min, then gel purify the ~450 bp fragment and quantify.

Gel Purification & DNA Quantification

Used the Sigma GenElute Gel Extraction kit. Imaging the gel showed a strong band at just smaller than 500 bp (no picture taken). DNA concentration is measured at 29.5 ng/µL, 260/280 is 2.0.

Ligation

Amounts given are for a 5:1 insert:vector ratio. Can scale back if gel purification yield is low.

Reagent Treatment (µL) Neg. control (µL)
Insert DNA (35 ng) 1.2 0
Vector DNA (50 ng) 2.5 2.5
2x Roche Rapid Ligation Buffer 5 5
NEB T4 Ligase 1 1
H2O 0.3 1.5
Total 10 10

Incubate at room temperature for 10 minutes.

Transformation

Follow the traditional transformation protocol found here. I am leaving the plates on my bench at room temperature (taped) over the weekend.