Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/17

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Plasmid Miniprep of DBN001_pSB1A3 (2nd attempt) Main project page
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Overview

Due to low yield of plasmid from last attempt, am trying again from streaked plates made earlier.

Notes

Once again, liquid culture density is very low. Suspect it's because there's no actual plasmid present.

Followed the instructions in the Sigma Plasmid Miniprep kit.

DNA Concentration

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
DBN001_pSB1A3 colony 178.274.981.89
DBN001_pSB1A3 colony 259.954.372.14

Restriction Digest

Reagent Volume
DNA600ng (7.7 - 10 µL)
10x FastDigest Buffer3
EcoRI1
SpeI1
Water17.3 - 15
Total30

Digest for 10 minutes at 37°C.

Gel Electrophoresis

Lost most of sample 1 while pipetting into the lane. Bands will probably be very faint.

Expected size fragments from EcoRI/SpeI digestion: 2132, 224

Image:2015-04-17_DBN001_pSB1A3_digest_annotated.png

Still no trace of plasmid present. Going to assume cloning did not work. Will try again next week with fresh phosphatase & ligase, using dephosphorylated backbone as another control.


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