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

Plasmid Miniprep of DBN001_pSB1A3

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Overview I'll be taking the two colonies from the sample plate and growing them up for plasmid miniprep. Notes 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 No trace of plasmid, just a smear of (presumably) genomic DNA. Will have to repeat plasmid prep and hope for a higher yield.