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Published 5 April 2014

Procedure of test for the presence of a pathogen in a wash water potato by PCR

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Here, you have three files of procedure of test for the presence of a pathogen in a wash water potato by PCR in word, in pdf, and the ppt for the presentation of this experiment.

Word - 54.5 kb
Test for the presence of a pathogen in a wash water potato by PCR - Protocol in word
PDF - 51.3 kb
Test for the presence of a pathogen in a wash water potato by PCR - Protocol in pdf
PowerPoint - 4.1 Mb
Test for the presence of a pathogen in a wash water potato by PCR - Practical training in ppt

Procedure of test for the presence of a pathogen in a wash water potato by PCR

A water wash sample was taken and was inoculated on LB (Luria-Bertani) culture medium.
Subsequently handling will be made from isolated colonies present on this medium.

1. DNA Extraction

1.1. Pipet 50μL of sterile distilled water in a sterile microtube (P1).

1.2. Take a bacterial colony with a calibrated 1μL sterile handle.
1.3. Make a suspension with the bacterial colony in the microtube (P1).
! Be careful to obtain a homogenous suspension!

1.4. Put the microtube (P1) in a water bath at 100°C during 1 minute.

1.5. Put the microtube (P1) in a centrifuge.
1.6. Centrifuge the microtube (P1) 5 minutes at 4500 rpm.

1.7. Remove the tube and transfer the supernatant containing the DNA into another sterile microtube (P2).

2. Preparation of the PCR mix (M)

2.1. Insert in a sterile microtube :
- 90 µL sterile distilled water
- 50 µL buffer 10X
- 28 µL MgCl2 25mM
- 28 µL dNTP 0,2µM
- 4 µL Taq polymerase

2.2. Freeze the tube if PCR is not done during the day.
2.3. At the moment of use, thaw if necessary and then centrifuge quickly.

! The PCR mix is already prepared !

3. DNA Amplification

3.1. In a microtube (A) for PCR pipet 10µL DNA solution from the microtube (P2).
3.2. Add 20µL PCR mix (M).
3.3. Add 10µL of each primers metkRPECTO and metkFPECTO or 16SR and 16SF.
It is possible to use both couples of primers as the same time. Volumes of primers are then 5μL.
3.4. Quickly Centrifuge microtube (A).

3.5. Place the microtube (A) in the thermocycler. Locate its position.

3.6. Perform two controls by repeating steps 3.1. to 3.5. by replacing the DNA solution (P2) by:
- Water for the negative control (C-)
- DNA solution Pectobacterium atro-septicum for the positive control (C+)

3.7. Set the thermocycler :
- 96°C, 5min
- 40 cycles :
— 94°C, 30s
— 58°C, 30s
— 72°C, 50s
- 72°C 5min
- storage at 4°C
3.8. Start the amplification.

4. Electrophoresis of DNA fragments

4.1. In three sterile microtubes (E1, E2, E3), place 10μL of PCR solution (A, C-, C +).
4.2. Add 2μL loading buffer (X) and stir.

4.3. Take the electrophoresis gel and remove protection.
4.4. Rinse deposit wells with demineralized water. Tilt the cassette to move excess fluid to the edge and then pat dry.

4.5. Place in a well, 5 µL of the test solution E1.
4.6. Place in two next wells, 5 µL controls E2 and E3.
4.7. Place in a fourth well 5µL solution of molecular markers (W)

! One electrophoresis gel will be used for 3 groups (12 wells) !

4.8. Start migration for ten minutes at 220V.
4.9. Stop the migration when the first strips of the solution of molecular markers reach the end of the gel.

5. Analyse your results