Week 5

7-6
The idea is to purify the plasmid that was inserted into competent Ecoli cells. We know they took up the plasmid because WT ecoli are not kan resistant, but all of the harvested bacteria was grown on LBKan plates. Competent Ecoli was plated to verify the plasmid was taken up.
Protocol for Plasmid DNA Purification:
1. Resuspend pelleted bacteria cells in 250µl Buffer P1 and transfer to a microcentrifuge tube
2. Add 250µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times
3. Add 350µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
4. Centrifuge for 10 min at 13,000rpm
5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting
6. Centrifuge for 30-60seconds and discard the flow-through
7. Wash the QIAprep spin column by adding .5ml Buffer PB and centrifuging for 30-60seconds. Discard Flow-through
8. Wash QIAprep spin column by adding .75ml Buffer PE and centrifuging for 30-60seconds
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
10. Place the QIA prep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50µl Buffer EB, let stand for 1 min, then centrifuge for 1 min.

DNA was then treated with .25µl Pst1, .25µl Hind3, 3.5 µl dH2O, 4µl plasmid, 1µl 10x Buffer 2, 1µl 10x BSA then incubated in the 37C water bath for 1 hour.

DNA was ran on a normal 1% gel and all bands were extremely faint except for the standard . Also none of the bands showed any discontinuity, indicating that genomic DNA managed to find it's way into the solutions. This is possibly a failure of precipitation or centrifuging, thus I will repeat the procedure tomorrow.

7-7
See above. The results from the previous day were replicated almost exactly. We believe it could be something with the restriction enzyme used; will investigate further.

7-8
Pst1 and Hind3 both tested individually with strain E3E along with a control with no enzyme added. All were run on a gel, which showed that Pst1 was cutting up the entire genome, whereas the other two both seemed to have a band as a part of the digest.

To test which enzymes are best, we will be cutting 3 different strains with BamH1, EcoR1, and Zho.