Objective
- Gel purify double digests from yesterday
- Transform ligation reactions from yesterday w/ non-ligated counterparts
- Re-plate transformations from 7/25
- Start midiprep cultures from streaked plates that grew overnight
- Perform ligation on double-digested plasmids and insert (from today's samples)
Bench work
- Phosphatase reaction with double-digested plasmids from yesterday
- → added 2μL antarctic phosphatase to the 100μL samples
- 2 Gels with the double digested samples:
- Gel 1: Taped 5 + 3 lanes together for two aliquouts of BSA PCR product → added 100μL to the gel with 1 ladder lane
- Gel 2: Taped 6 lanes together for each of the plasmid samples → Got 95% of the samples into the gel + 1 ladder lane
- Followed protocol for Promega Wizard® SV Gel and PCR Clean-up system for the two gels
- Picked 3 colonies from each of the glycerol stock-streaked plates and put into 3mL cultures of LB + Amp for the day
- Inoculated 100mL cultures at end of day + Amp
- Transformed 4 ligation test samples:
- ("PNL") pTXB1 digested with EcoRI, BamHI, and antarctic phosphatase
- ("PL") pTXB1 digested with EcoRI, BamHI, and antarctic phosphatase...treated with T4 DNA ligase
- ("NPNL") pTXB1 digested with EcoRI, BamHI
- ("NPL") pTXB1 digested with EcoRI, BamHI...treated with T4 DNA ligase
Results
DNA quantitation from gel purification
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Gels
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AU CHEM-570 Lab Prep
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