| 1. PCR Reaction: PCR LacI/LAMBDAcI and TetR/p22mnt dual promoters:' PCR'ed these two dual promoters to remove RBS so that it can be submitted to iGEM as a single part. Follow the table below:
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| PRIMER MIXES below...
| Dual Promoters | prim 1F | prim 2R | H20
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| LacI/LAMBDAcI | 1.5uL | 1.5uL | 12.0uL
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| TetR/p22mnt | 1.5uL | 1.5uL | 12.0uL
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| PCR: Dual Promoters below...
| Parts | 5x HF Buffer | dNTP's | primer mix | DNA template | phusion hot start polymerase | H20 | Total
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| Lac/LAMBDA | 40.0uL | 4.0uL | 10.0uL | 40.0uL | 2.0uL | 104.0uL | 200.0uL
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| TetR/p22mnt | 40.0uL | 4.0uL | 10.0uL | 29.0uL | 2.0uL | 115.0uL | 200.0uL
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| L/L control | 10.0uL | 1.0uL | 2.5uL | 0 | 0.5uL | 36.0uL
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| T/P control | 10.0uL | 1.0uL | 2.5uL | 0 | 0.5uL | 36.0uL
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| 2. Different PCR reaction: Used PCR to produce more lambdacI gene with the RBS ligated to the 5' end Once the PCR procedure had been successfully completed, the PCR product (LAMBDAcI) was then used in the double digest along with RFP.
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| 3. Double Digest: Digested two things only - LAMBDAcI and RFP. Used higher concentrations of parts' DNA to attempt a better spec'ing result. Incubate digest products for 2 hours. Heat inactivate enzymes for 15 minutes in a 65C water bath. Follow the table below:
| Parts | 10x Buffer | BSA | DNA | RE 1 | RE 2 | H20
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| LAMBDAcI | 5.0uL | 0.5uL | 40.0uL | Xba1, 1.0uL | Pst1, 1.0uL | 2.5uL
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| RFP | 5.0uL | 0.5uL | 18.0uL | Xba1, 1.0uL | Pst1, 1.0uL | 24.5uL
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3. Gel electrophoresis: Ran a gel to check if any DNA or correct DNA is in digested products. The gel confirmed presence of digest products (LAMBDAcI and RFP) of the correct size Lanes (L -> R) are 1kb ladder, two lanes of lambda cI digest, two lanes of RFP digest, and a second 1 kb ladder. The three bands in the RFP sample are believed to be undigested base vector + RFP, plasmid backbone, and the RFP gene. Digested Lambda cI gene and RFP gene (lowest bands in picture) were cut out for purification.
 Electrophoretic gel run on 7.29.08 |
| 4. Gel purified: Purify the DNA bands out of the gel by following previously performed purification techniques to 'purify' or extract the DNA from the gel.
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| 5. Ligation: Ligate (1) RFP + T/P + BV and (2) LAMBDAcI + TetR promoter + BV. In #1, the BV and TetR/p22mnt dual promoters are already there - are just ligating into that the RFP. In #2, the BV and TetR promoter are already there - are just ligating the LAMBDAcI onto that. After ligated, allow to incubate 30 minutes then heat shock enzyme for 15 minutes @ 65C in a water bath. Then transform into competent cells. Follow the table below:
| Parts | 10x Buffer | H20 | Base Vector 1 | Base Vector 2 | T4 DNA ligase | Total
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| RFP + T/P + BV | 6.0uL | 0 | 4.0uL | 44.0uL | 6.0uL | 60.0uL
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| LAMBDAcI + Tet promoter + BV | 6.0uL | 0 | 4.5uL | 23.5uL | 6.0uL | 60.0uL
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| 6. Transform ligated products into competent cells: Allow to incubate O/N @37C with shaking at 220rpm's.
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