| 1. Ran the terminator digest out on a gel: After which we were unable to see a band the appropriate length, so we simply cut out the gel where we expected the band to be (terminator is very small - so hard to see). Gel purified - then used this in our next ligation. When spec'ed the purified gel terminator product, there were 2.5ngram/uL of DNA concentration.
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| 2. Currently have two parts that are base vector-promoter-gene, and one that is base vector-promoter: We are currently ligating the terminator to the two parts that have vector-promoter-gene.
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| The base vector-promoter-gene constructs are: TetR promoter:Lambda cI and Tet/p22 promoter:RFP.
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| The base vector-promoter construct is: Lambda cI/Lac promoter
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| 3. Digest Rxn: Digested Terminators, Lac/LAMBDAcI, and GFP. Follow the table below:
| Parts | 10x Buffer | BSA | H20 | DNA | RE 1 | RE 2 | Total
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| Term 2 | 5.0uL | 0.5uL | 39.5uL | 3.0uL | 1.0uL, Xba1 | 1.0uL, Pst1 | 50.0uL
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| Term 4 | 5.0uL | 0.5uL | 28.5uL | 14.0uL | 1.0uL, Xba1 | 1.0uL, Pst1 | 50.0uL
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| Lac/LAMBDA A | 5.0uL | 0.5uL | 8.0uL | 34.5uL | 1.0uL, Spe1 | 1.0uL, Pst1 | 50.0uL
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| Lac/LAMBDA B | 5.0uL | 0.5uL | 16.0uL | 26.5uL | 1.0uL, Spe1 | 1.0uL, Pst1 | 50.0uL
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| GFP | 5.0uL | 0.5uL | 40.5uL | 2.0uL | 1.0uL, Xba1 | 1.0uL, Pst1 | 50.0uL
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