Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/29: Difference between revisions

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Load 15 µL of digest on the gel. Expected size fragments are 525, 1246, and 2536bp.
Load 15 µL of digest on the gel. Expected size fragments are 525, 1246, and 2536bp.


[[Image:2015-04-29_DBN004_pSB1A3_SpeI_ApaLI_digest_annotated.png]]
All twelve colonies are have size fragments at ~1200, ~500 and ~400bp. This doesn't match with an SpeI/ApaLI digestion of DBN004_pSB1A3, but it does match with plain pSB1A3 when digested with the same enzymes. My guess is the few colonies that survived Type IIS digestion/ligation were the ones that could excise the insert and retain the ampR gene in the plasmid.
I also didn't realize this until just now, but there's a BsaI cut site inside the AmpR gene. Should have checked that before ordering the DBN001 fragment and before amplifying the DBN002 & DBN003 fragments. Not sure how to proceed from here.


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Revision as of 17:12, 29 April 2015

Plasmid Prep & Validation of DBN004_pSB1A3 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Overview

After picking 12 colonies to streak and grow liquid cultures, I'm going to harvest them, perform plasmid minipreps, quantify DNA, and do a basic verification using restriction digest and gel electrophoresis.

Plasmid Miniprep

All twelve colonies grew in liquid culture. Samples were prepared using the Sigma GenElute Plasmid Miniprep kit.

DNA Concentration

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
1.1 108.1 2.76 1.95
1.2 140.5 11.25 1.95
1.3 161.1 1.54 1.92
1.4 146.0 2.89 1.94
2.1 149.9 0.79 1.93
2.2 185.7 24.56 1.88
2.3 193.9 21.42 1.90
2.4 110.9 1.89 1.89
5.1 149.3 8.35 1.91
5.2 148.3 0.37 1.88
5.3 142.3 17.26 1.88
5.4 123.6 1.00 1.91

Restriction Digest

Reagent Volume
DNA 600ng (5 µL)
10x FastDigest Buffer 3
ApaLI 1
SpeI 1
Water 20
Total 30

Digest for 10 minutes at 37°C.

Gel Electrophoresis

Load 15 µL of digest on the gel. Expected size fragments are 525, 1246, and 2536bp.

All twelve colonies are have size fragments at ~1200, ~500 and ~400bp. This doesn't match with an SpeI/ApaLI digestion of DBN004_pSB1A3, but it does match with plain pSB1A3 when digested with the same enzymes. My guess is the few colonies that survived Type IIS digestion/ligation were the ones that could excise the insert and retain the ampR gene in the plasmid.

I also didn't realize this until just now, but there's a BsaI cut site inside the AmpR gene. Should have checked that before ordering the DBN001 fragment and before amplifying the DBN002 & DBN003 fragments. Not sure how to proceed from here.


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