Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/09

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Overview

DBN011 ligation results:

  • Negative control: 8 colonies
  • Treatment: 18 colonies

Going to pick 4 colonies from the treatment plate and proceed with streaks & liquid cultures.

To do:

  • plasmid prep
  • DNA quantification
  • Restriction digest, HA1_v0120 with E&S (106 bp)
  • Restriction digest, DBN011 with E&X (5769 bp)
  • DNA quantification
  • ligation
  • transformation

Plasmid preps

  • HA1_v0120 1: 24 ng/µL
  • HA1_v0120 2: 7 ng/µL
  • DBN011 1: 124 ng/µL
  • DBN011 2: 106 ng/µL
  • DBN011 3: 27 ng/µL
  • DBN011 4: 96 ng/µL

Restriction Digests

HA1 (E&S):

Reagent Volume (µL)
DNA25
FD 10x GRN3
EcoRI1
Spe11
Total30

DBN011 (E&X):

Reagent Volume (µL)
DNA25
FD 10x GRN3
EcoRI1
XbaI1
Total30

Incubate at 37 °C for 10 minutes, then gel purify. HA1 part is 106 bp, DBN011 part is 5769 bp.

Gel Extraction Results

HA1 part looks good, but very faint band. DBN011 1, 2, and 4 look good. Proceeding to extract 1 and 2.

  • HA1 (E&S digested): 4 ng/µL, 260/280 high (~3)
  • DBN011 1 (E&X digested): 18.1 ng/µL
  • DBN011 2 (E&X digested): 13.6 ng/µL

Ligation

Reagent Volume (µL) Negative
Vector (DBN011 1) (50 ng)2.82.8
Insert (HA1) (10:1)2.30.0
10x buffer1.01.0
ligase1.01.0
H2O2.95.2
Total10.010.0

Incubate at room temperature for 10 minutes, then take 1 µL of reaction volume and use for quick transformation. Count colonies tomorrow.



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