Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/05/05

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Overview

All six liquid cultures grew as expected. Harvested cells and performed a plasmid miniprep for each. The BioBrick cloning constructs ( from KAH57 & KAH104) I'm naming DBN006.

DNA Concentration

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
DBN006 1 383.4 3.20 1.94
DBN006 2 671.2 11.91 1.93
DBN006 3 298.6 7.32 1.94
KAH57 259.3 3.25 1.93
KAH104 293.7 44.45 1.91
pMaxGFP 549.5 7.52 1.92

Will verify via restriction digest tomorrow.

Restriction Digest (05/06)

Part Conc. (ng/µL) Volume for 600ng (µL) Volume water (µL)
DBN006 1 383.4 1.6 23.4
DBN006 2 671.2 0.9 24.1
DBN006 3 298.6 2.0 23.0
KAH57 259.3 2.3 22.7
KAH104 293.7 2.0 23.0
Reagent Volume (µL)
DNA + water 25
10x FD Buffer (Green) 3
EcoRI 1
SpeI 1
Total 30

Incubate at 37°C for 10 minutes, then run total volume on the gel.

Digest Results

2015-05-06 expected bands.png 2015-05-06 DBN006 KAH57 & KAH104 digests annotated.png

Colonies 1 and 3 contain inserts of the expected size.