IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2014/07/30

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Week 7 Day 3

- Picked 1 white colony from 29/07/14 transformed dig-lig plates and added to 5 ml LB + 5 μl Ampicillin and incubated at 37 °C also added to 50 μl of PCR Master Mix containing:

1 μl DNTPs

2 μl Primer 1

2 μl Primer 2

5 μl Buffer

2 μl MgCl2

0.5 μl BIOTAQ Enzyme

37 μl Water

- PCR programme:

Step 1 : 95 °C 5 min Step 2 : 95 °C 1 min Step 3 : 54 °C 1.5 min Step 4 : 72 °C 1 min Step 5 : 72 °C 5 min

Steps 2, 3, 4 repeated x 34 times

- Run on a 1 % agarose TAE gel at 100V. Each well loaded with 2 μl of loading dye and 20 μl of DNA. Two 1kb ladders loaded.

- DNA mini prep of cultures from 29/07/14 (DNA 2.0 level 0's and PRO, CDS, TER) (2.5 mls)

- Glycerol stocks made from 1 ml of O/N culture.

- Transformations of DNA iGEM #2-19 and Vectors #1-4 intro E.coli to generate more DNA.


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