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