IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/06: Difference between revisions

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==PCR parts of Agr operon==
==PCR parts of Agr operon==

Revision as of 09:40, 19 August 2008

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Result from transformation

Check Vectors

- Plasmid miniprep for ECE176 (from 05/08 LB stock and from 04/08 LB stock with a second inoculation)

We want to check uncut plasmid, single and double digest for ECE147, ECE149, ECE150, ECE153, ECE162, ECE172, ECE176. In order to have enough DNA (to see the bands), we will add 1μg of DNA to make single digest, and wr will incubate during 1h (instead of only 10min).

  • Nanodrop
Vetor 260/280 μg/mL
ECE147 1.67 50.8
ECE149 1.66 51.9
ECE150 1.78 107.0
ECE153 1.68 31.2
ECE162 1.56 33.1
ECE172 (04/08) 1.64 22.8
ECE172 (05/08) 1.89 213.1
ECE176 (04/08) 1.89 111.1
ECE176 (05/08) 1.83 96.2

We made singe digest for the samples from 05/08 only.

- Double digest (1 hour of incubation in water bath at 37°C)

- Gel

  • Results

Our supercoiled ladder was very bad, so it was impossible to conclude for uncut vectors.

- ECE147, ECE 150, ECE153, ECE 176 single digest : ok

- ECE149 : problem, 2 cutting sites for PstI (only one in the sequence)

- ECE162 : only one band whereas SalI should have 2 cutting sites

- ECE172 : wrong sizes!!!

- Double digest for ECE176 : ok!

- Double digest for ECE 150, 149 and 147 : only one band!!!

There may be a problem when we do the double digest by adding two single digest. We will try again with a direct double digest.



PCR parts of Agr operon

We received primers for Agr A,B,C, and D today. We must PCR each individual part out of the previous BioBricks (I746001 &I746101) in order to put our bacillus RBS on them.

I was concerned that the region of most homology would be the BioBrick prefix and suffix itself, so I first digested the DNA with Xbal and SpeI.

For some reason the digestion didn't work that well, so I've decided to go ahead and use the uncut BioBrick DNA as template for my reactions.

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