SITE-DIRECTED MUTAGENESIS BY PCR AMPLIFICATION

 The first step in the mutagenesis of the GFP gene is to methylate the plasmid which carries the gene. This plasmid, pGLO, has been propagated in E. coli and then purified. We have done the methylation for you due to time constraints but the procedure is listed below. The methylase used transfers methyl groups from S-adenosylmethionine to cytosine residues occurring next to guanine. When DNA is methylated in this way, and then transformed into a wild-type strain of E. coli the DNA will be degraded. Thus after amplification of the plasmid with mutagenic primers, resulting in a mutagenized plasmid that is not methylated, only this new plasmid DNA will be replicated in the cell.

Methylation reaction

Mix together:

100 ng of the pGLO plasmid

1.6 μl of 10X methylation buffer

1.6 μl of 10X S-adenosylmethionine (SAM)

1 μl DNA methylase

water to 16 μl total volume

Incubate at 370C for 1 hour.

YOU WILL BEGIN AT THIS STEP

Mutagenesis reaction

Mix together:

4 μl methylated plasmid

1 μl of pGLO primer mix

45 μl PCR Supermix High Fidelity

Cycling parameters:

94 C for 2 min.

94 C for 30 sec.

55 C for 30 sec.

68 C for 6 min.

back to step 2 for 20 more cycles

68 C for 10 min.

After amplification is complete remove 17 μl of the PCR reaction to a clean microfuge tube, add 3 μl of tracking dye, and run on an agarose gel to verify amplification. Next, 4 μl of the reaction is transformed into E.coli DH5 and plated on a rich media containing ampicillin and arabinose. The ampicillin selects for the plasmid and the arabinose induces the transcription of the GFP gene. It is important to keep the cells on ice and chilled until the heat shock in step 4. Never vortex competent cells.

1. Put 4 μl of each ligation reaction in a sterile 1.5 ml tube and place it on ice. Thaw competent cells on ice.

2. Transfer 50 μl of competent DH5α into each tube and gently mix by tapping the tube.

3. Incubate tubes on ice for 15 min.

4. Heat-shock the cells for 3 min. at 42°C.

5. Return tubes to ice for 2 min.

6. Add 900 ml LB broth and incubate at 37°C for 1 hour.

7. Spin culture, remove most of the media, resuspend pellet in remaining media and plate on LB + 100 μg/ml Ampicillin + 0.6% Arabinose

ISOLATION OF GENOMIC DNA FROM GRAM NEGATIVE BACTERIA

You will be given a 1.5 ml microcentrifuge tube containing a pellet of Escherichia coli, strain BW30270. This strain is a K-12 or laboratory strain of E.coli. The pellets are made up of cells from 3 ml of overnight growth in a rich medium.

1. Add 600 μl of Nuclei Lysis Solution. Gently pipet up and down until the cells are resuspended.

2. Incubate at 80°C for 5 minutes to lyse the cells; then cool to room temperature.

3. Add 3μl of RNase Solution to the cell lysate. Invert the tube 2–5 times to mix.

4. Incubate at 37°C for 30 minutes. Cool the sample to room temperature.

5. Add 200 μl of Protein Precipitation Solution to the RNase-treated cell lysate.

6. Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution with the cell lysate, be sure that the two solutions have completely mixed. Do not over-vortex or you risk shearing the chromosome.

7. Incubate the sample on ice for 5 minutes. Centrifuge at 13,000 rpm for 10 minutes.

8. Transfer the supernatant containing the DNA to a clean 1.5ml micro-centrifuge tube containing 600μl of room temperature isopropanol. Do not carry over any flecks of the precipitate.

9. Gently mix by inversion until the thread-like strands of DNA form a visible mass. If you do not see threads but a general whitish appears mix well and continue.

10. Centrifuge at 13,000 rpm for 5 minutes.

11. Carefully pour off the supernatant and drain the tube on clean absorbent paper. Add 600 μl of room temperature 70% ethanol and gently invert the tube several times to wash the DNA pellet.

12. Centrifuge at 13,000 rpm for 2 minutes. Carefully aspirate the ethanol from the tube with a pipet. Be careful not to suck up your pellet.

13. Drain the tube on kimwipes and allow the pellet to air-dry for 10–15 minutes.

14. Add 100μl of sterile water to the tube and rehydrate the DNA.