Team:Panama/14 October 2010

October 14

Prepared Ampicillin stock (50 mg/ml)

We did a miniprep on the 11 colonies that grew in liquid LB medium with Amp. (The method we used was without columns because the kit ran out). Then, we ran a gel of 1% agarose to see if we could see a difference in the size of the linear band of the plasmid to be able to determine if it contains the insert (Rh1ab) or not.

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Miniprep Protocol:

1. Inoculate 3 ml LB medium (containing antibiotic) with a bacterial clone, culture with vigorous shaking at 37 degrees for 14-16 hours.

2. Aliquot 2 x 750 ul culture into microcentrifuge tube, harvest bacteria by spinning at 12000rpm for 1 minute. Aspirate supernatent. Add an additional 750 ul culture media, respin and aspiate supernatant.

3. Resuspend bacterial pellet by complete vortexing in 110ml resuspension buffer (100 ul solution A (25 nM Tris-HCl, pH 8.0, 10 mM EDTA) + 10 ul RNase A). The bacteria should be completely resuspended- no clumps should be visible. Solution A is stored in the refrigerator. RNAse is in the -30C freezer.

4. Add 100 ul freshly prepared lysis buffer (50 ul 400 mM NaOH, 50 ul 2% SDS) and mix gently by flipping.