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