Minnesota/25 July 2008

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Revision as of 20:49, 25 July 2008 by Emartin9808 (Talk | contribs)
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1. No growth on plates: Experiments on 07-24-08 failed to show growth on Kanamycin resistant plates.
2. Proposed Schedule for today: 10:40-12:40: BV Digest...12:40-12:55: Restriction Enzyme inactivation...1:15-1:45: BV Dephosphorylation...1:45-1:55: Phosphatase enzyme inactivation...2:15-2:45: Ligation @ 16C...2:45-3:00: Ligase enzyme inactivation...3:00-3:45: Transformation...5:45: Plating on kan. res. plates.
3. 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
4. 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
5. 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).