Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/16

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I'll be taking the two colonies from the sample plate and growing them up for plasmid miniprep.


Both colonies grown in liquid culture are normal colored (not red). Cell density appears low though, with lots of sticky filaments. Indication that they were not subjected to continuous shaking overnight.

Also, for future reference: when performing digestion/ligation, set up a control plate using linearized vector treated with phosphatase. Presence of colonies on this plate indicates that the phosphatase step did not completely work.

DNA Concentration

Sample ID Conc. (ng/µL) St Dev (ng/µL)
DBN001_pSB1A3, colony 1 56.7 2.6
DBN001_pSB1A3, colony 2 54.0 0.2

Restriction Digest

Reagent Volume
DNA 600ng (12 µL)
10x FastDigest Buffer 3
EcoRI 1
PstI 1
Water 13
Total 30

Digest for 15 minutes at 37°C.

NOTE: Should've used SpeI, not PstI. Oh well, won't make much of a difference for analysis of the sequence.

Gel Electrophoresis

Expected size fragments from EcoRI/PstI digestion: 2114, 189, 53

2015-04-16 DBN001 pSB1A3 digest annotated.png

No trace of plasmid, just a smear of (presumably) genomic DNA. Will have to repeat plasmid prep and hope for a higher yield.