Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/06
|Plasmid Miniprep for pTet-EsaI-GFP_pSB1A3||Main project page|
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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.
Plasmid concentration (average, n=2):
37°C for 20 minutes.
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):
Expected size of fragment is 244bp, which matches the band size on the gel.