Induction of Expression for Exogenous Gene

1. Grow cells overnight in LB ampicillin.
2. Get an OD595 of a 50 fold dilution of the overnight culture. We will use the plate reader to do this. The final volume in a well should be 200 µl and make sure you have one blank well of LB ampicillin.
3. Calculate the OD595of your overnight culture and set up a dilution in 2 ml of fresh LB amp so that the OD595 of this new dilution will be 0.2.
4. Grow the cultures until the OD595 reaches 0.5 (doubling time of about 20 minutes).
5. Add IPTG to a final concentration of 0.4 mM (stock is 100 mM) and incubate for 1 hour.
6. Spin cells for 2 minutes in microfuge and transfer to a small microfuge tube.
7. Resuspend the pellet in 200 µl of cold lysis buffer (50 mM Tris-HCl, pH 8.0; 2 mM EDTA). Add lysozyme to concentration of 100 µg/ml and 0.1% Triton X-100.
8. Incubate this lysis reaction for 30 minutes at 30° C.
9. Add 100 µg/ml DNase** (stock solution is 10 mg/ml) to this reaction for 45 minutes at 37° C.
10. Spin lysed cells for 15 minutes in microfuge. Resuspend pellet in 20 µl of water and store at 4° C.

During incubations, different members of each lab group can push the protocol forward while the other one is working on the computer. The idea is to have both the computer analysis and the lab portion be pushed forward during the same lab period. Since only one person can work on a computer at a time to analyze his or her sequences, this should not present a problem.

**WARNING!! DNase is a nasty reagent that we CANNOT have spread around a lab where we spend most of our time working with, and protecting, DNA.

Therefore, use DNase only in the designated places.

Lysis Buffer with Lysozyme

• 500 µl 1 M Tris, pH 8.0
• 40 µl 0.5 M EDTA, pH 8.0
• 10 µl Triton X-100

Lab Schedule Outlined

Lab Schedule In Context of Research Project