840:153g:Projects/project23/2012/11/15

Cloning E.coli with the OmcF gene

• On Tuesday, we worked on the digestion of our plasmid DNA with the restriction enzymes SpecI and PstI and 10x Digestion Buffer. * We then purified the sample with the PCR purification kit to rid the sample of any additional parts.
• To check the approximate quantity of our samples, we ran a gel along with a Low Range Ladder to judge the amounts of each sample. After this we determine the appropriate concentrations to use during ligation to help aid that process.
• They were loaded into the well as follows:

1. 100bp ladder
2. 2x plasmid DNA
3. 2x plasmid DNA
4. 4μL Low Range Ladder
5. 2μL Low Range Ladder
6. 450bp 2x DNA
7. 450bp 1x DNA
8. 350bp 2x DNA
9. 350bp 1x DNA
10. 100bp Ladder

• Result: Wells are shown No. 10 - 1 above. Plasmid DNA is intact, and our fragment DNAs are in correct position. Well 9 is overrepresented slightly, and all other sample combinations are comparable. The samples are useable for transformation, but in differing ratios to find the most effective combination.

• We then ligated the samples with concentrations of 4:4, 0.5:7.5, and 7.5:0.5μL of plasmid DNA to our insert DNA (OmcF DNA). We also did 2 controls for each 450 and 350 DNA sample. One with no plasmid DNA and one with no insert DNA

• On Thursday, we continued to work on the transformation, by actually combining our ligated samples with 40μL of E.coli cells, icing, heat shocking, then icing the samples. We then added SOC and shook the samples for an hour at 37°C.
• We then plated our samples on Kanamycin solid media plates.