Plasmid Purification (QIAprep^® Spin Miniprep Kit)

1. 1-5ml bacterial overnight culture was pelleted by centrifugation at >8000rpm for 3 minutes at room temperature.

2. Pelleted bacterial cells were resuspended in 250µl of Buffer P1 and transferred to a microcentrifuge tube.

3. 250µl of Buffer P2 was added and mixed thoroughly by inverting the tube 4-6 times until solution becomes clear.

4. 350µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times.

5. The mixture was centrifuged for 10 minutes at 13000rpm in a microcentrifuge.

6. The supernatant was applied to a QIAprep spin column by pipetting.

7. The QIAprep spin column was washed with 500µl of Buffer PB by centrifugation for 1 minute and flow through discarded.

8. 750µl of Buffer PE was added to the QIAprep spin column and centrifuged for 1 minute and flow through discarded.

9. The QIAprep spin column was spun for an additional minute to remove residual wash buffer.

The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 50µl of H

O to the QIAprep spin column and centrifuging for 1 minute.

Overnight Cultures

1. Four colonies were picked from the desired plate.

2. For each colony, 5ml of fresh LB plus the appropriate antibiotics was inoculated and grown overnight at 37^oC.

SDS-PAGE and Western Blotting

1. 50µl of an overnight culture was used to inoculate 5ml of fresh LB containing the appropriate antibiotics and left to grow at 37^oC in a shaking incubator.

2. Once the OD₆₀₀=0.6-0.8, 1mM of IPTG was added to the culture and left to grow for a further 3 hours.

3. 1ml of each culture was taken and spun down in a microcentrifuge for 3 minutes.

4. Meanwhile, 950µl of 2x Laemmli sample buffer was added to 50µl of B-mercaptoethanol.

5. The supernatant was discarded from the samples and the pellet resuspended in 200µl of the above solution.

6. The samples were then boiled at 90^oC for 10 minutes.

7. Afterwards, they were spun down for 1 minute in a microcentrifuge.

8. At this stage, the samples could be stored at -20^oC or ran on a gel immediately.

9. Two gradient gels consisting of a 12% separating gel and a stacking gel were prepared and the samples ran at 100V until proteins reach end of the stacking gel at which point the voltage was increased to 200V for 20 minutes.

10. One gel was stained using Instant Blue and the other was used to transfer the proteins onto a membrane.

11. After the proteins had been transferred onto the membrane, the membrane was blocked in 5% Marvel milk overnight at 4^oC.

12. The next day, the membrane was washed 3 times in 1xTBST buffer and then suspended in 10ml of 1xTBS buffer to which the primary anti-His antibody was added and left for one hour. The protein ladder was cut off before adding the primary antibody.

13. The membrane was then washed 3 times in 1xTBST buffer for 5 minutes and then suspended in 10ml of 1xTBS buffer, to which the secondary antibody was added and left for one hour.

14. The membrane was then washed in 3x10ml 1xTBST buffer for 5 minutes.

15. The blot was then soaked in developing solution for 5 minutes before being wrapped in a plastic cover aligned with the protein ladder that was cut off at step 12.

Once in the developing room, the film was exposed to the blot for 30 seconds, 2 minutes and 5 minutes and then placed in the film developer.