Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/06

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Overview

The pTet-EsaI-GFP_pSB1A3 plasmid (pSB1A3-GFP for short) is a cloning plasmid with Tet promoter followed by EsaI and GFP. Full annotated sequence can be found here. I've been trying to use this plasmid as the backbone for insertion of the homology arms and cloning region for HPK-CFP insertion, but have not had any success yet. I also have not observed any green fluorescence in colonies transformed with this plasmid. Ran out of plasmid and needed to make more.

  • DH5α-Turbo was transformed with pSB1A3-GFP and grown on LB-Amp plates over Thursday night. Liquid cultures and backup plates were started on Friday. Cells were harvested by centrifugation on Saturday and stored at -20°C until today.
  • Performed a plasmid miniprep on the cells, followed by nucleic acid quantification using the BioTek Take3 plate reader. Plasmid is stored in molecular biology-grade water.

Plasmid concentration (average, n=2):

Sample ID 260/280 ng/µL
pSB1A3 colony 1 1.9115 341.6 +/- 3.6
pSB1A3 colony 2 1.9115 681.3 +/- 47.1

pTet-EsaI-GFP_pSB1A3 digestion:

Reagent Volume (µL)
FD 10x Buffer 2
Water 15
EcoRI 1
PstI 1
DNA 1 µg (1-3 µL)

37°C for 20 minutes.

Gel image:

2015-04-07 pTet-EsaI-GFP pSB1A3 gel annotated v2.png

gBlock PCR verification

The gBlock containing homology arms and IIS restriction site was shipped with only 200ng total; therefore it was necessary to perform PCR to make more copies of the gBlock. PCR details can be found here.

PCR product concentration (average, n=2):

Sample ID conc. (ng/µL) stdev (ng/µL)
gBlock 399.2 11.74

Gel image: 2015-04-07 gBlock PCR product gel annotated.png

Expected size of fragment is 244bp, which matches the band size on the gel.