Minnesota/25 July 2008

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Revision as of 20:44, 25 July 2008

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1. No growth on plates: Experiments on 07-24-08 failed to show growth on Kanamycin resistant plates.

2. Double Digest: Redo double digest of Base Vector. Follow the table below:

Parts	10x Buffer	BSA	H20	Insert DNA	RE 1	RE 2
Base Vector	5.0uL	0.5uL	39.5uL	3.0uL	1.0uL, EcoRI	1.0uL, Pst1

3. Redoing ligation: Using left over double digest from 07-24-08, But increase the amount of ligase in the reaction and performing the ligations @ 60C which is the optimal temperature for the ligase.

Parts	10x Buffer	H20	Base Vector	Insert DNA	T4 DNA Ligase	Total Volume
BV + Tet1	4.0uL	14.0uL	1.0uL	17.0uL	4.0uL	40.0uL
BV + Lac2	4.0uL	16.0uL	1.0uL	15.0uL	4.0uL	40.0uL
BV + Lac/LAMBDA	4.0uL	18.0uL	1.0uL	13.0uL	4.0uL	40.0uL
BV + Tet/p22	4.0uL	14.0uL	1.0uL	17.0uL	4.0uL	40.0uL

4. Redoing transformations: Redo with new ligated products by adding much less DNA/plasmid to the TOP10 Cells, b/c if put too much ligated DNA then the cells do not transform properly (as stated by iGEM troubleshooting). Add transformed products to 250uL of SOC broth, since this type of broth is ideal for TOP10 Cells. Warm SOC broth in tubes in an incubator before adding transformed products (product + TOP10 Cells).

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