PCR Analysis of Agrobacterium

This protocol was contributed by Phil Wigge (Salk Institute).

Selection of transformed Agrobacterium can be ambiguous, because it normally takes several days for resistant colonies to appear. Since transformation of Arabidopsis is a relatively long-winded process, it is a good idea to verify the presence of the T-DNA plasmid in the Agrobacterium strain before proceeding with transformation experiments. Similarly, when retrieving Agrobacterium from a frozen stock, it is advisable to verify the presence of the correct T-DNA. For PCR, try to choose primers that identify the construct uniquely (e.g., for fusion of the CaMV 35S promoter to your favorite gene, use one primer in the promoter and another in the gene of interest).

Procedure

1. Dilute 1 pi of Agrobacterium-satuxated culture in 10 pi of sterile distilled water.

2. Cover the culture with a layer of mineral oil, and incubate it for 3 minutes at 100°C in a thermocycler.

If using a thermocycler with a heated lid, the mineral oil may be omitted.

3. Pause the program at 85°C while making up the PCR mix.

1 Ox reaction buffer

2 ill

2 mm dNTPs

4 fi.1

100 mm MgCl2

0.4 |il

20 jlim primer A

1 Hi

20 JIM primer B

lpl

Taq polymerase

0.25 \xi

double-distilled water

0.35 fil

4. Add 9 pi of PCR mix to each Agrobacterium sample. Mix gently.

5. Carry out a total of 35 cycles using standard PCR conditions.

6. Analyze the PCR products on an agarose gel.

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