SeM PCR protocol flow sheet

1. Hazard Assessment

   To protect yourself from any possible hazards associated with this task wear eye protection. You should also wear latex, nitrile, or vinyl gloves and a lab coat with long sleeves. To protect your legs and feet wear closed shoes and long trowsers. Do not wear sandles, shorts or a short skirt. Wash your hands before eating and when leaving the laboratory. You should review the MSDS for any chemical used in this procedure. In case of a spill with a toxic chemical remove all contaminated clothing and wash affected areas with copious quantities of water. Check location of the nearest safety shower. Eyes should be washed copiously for 15 minutes.

   This protocol is based on Kelly et al. 2006.

2. Reagents

   Reagent	Amount	Separate SOP?	Ready
   Lysozyme solution (pH 6.0)	8.2 mL	Yes	____
   Proteinase K (20mg/mL)	0.5 mL	Yes	____
   Wash Solution Concentrate	80 mL	Yes	____
   Buffer PB	20 mL	Yes	____
   Buffer PE	30 mL	Yes	____
   Buffer EB (10 mM Tris-Cl, pH 8.5)	2.0 mL	No	____
   Lysis Solution C for cell lysis	20 mL	No	____
   Column Preparation solution	60 mL	No	____
   Ethanol (95%) for dissolution of all elements except for DNA	8.0 mL	No	____
   Wash Solution 1	50 mL	No	____
   Elution Solution (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0)	8.0 mL	No	____
   Forward Primer	40.0 µL	No	____
   Reverse Primer	40.0 µL	No	____
   Vent DNA Polymerase	8.0 µL	No	____
   dNTP	40.0 µL	No	____
   Distilled Water	0.6 mL	No	____
   Sodium Acetate (3.0M)	0.4 mL	No	____
   Comments:
   Materials			Ready
   Pipette tips			____
   Microcentrifure tubes			____
   GenElute Nucleic Acid Binding Columns in tube			____
   Collection Tubes, 2.0 mL capacity			____
   55 C water bath			____
   37 C water bath			____
   Pipette tips			____
   1.5 mL microcentrifuge tubes			____
   Microcentrifuge			____
   QIAquick spin columns			____

   Comments:

3. Bacterial Preparation and Suspension

   Prepare a solution of Mutanolysin and Lysozyme and label	____
   Suspend a single S. equi colony in 200µL of Mutolysin/Lysozyme solution	____
   Incubate the solution for 1 hour and 37 C	____

   Comments:

4. Cell Lysis

   Add 20 µL of Proteinase K to solution	____
   Add 200 µL of Lysis solution C to solution	____
   Vortex solution for 15 seconds	____
   Incubate at 55 C for 10 minutes	____

   Comments:

5. DNA Isolation

   Add 500 µL of column preparation solution to each GenElute Miniprep Binding Column seated in 2 mL collection tube	____
   Centrifuge at 12,000 x g for 1 minute	____
   Discard Elute	____

   Comments:

6. Binding preparation

   Add 200 µL of ethanol to the lysate	____
   Vortex solution for 10 seconds	____

   Comments:

7. Lysate

   Add solution to the prepared binding column*	____
   Centrifuge at 6500 x g for 1 minute	____
   Discard collection tube with eluate and place column in a new 2mL collection tube	____

   *Use wide tip pipet in order not to splice DNA
   Comments:

8. Wash

   Add 500 µL of wash solution 1c	____
   Centrifuge at 6500 x g for 1 minute	____
   Discard collection tube with eluate and place in a new 2 mL collection tube	____
   Add 500 µL of wash solution to column*	____
   Centrifuge at maximum speed for 3 minutes to dry column**	____
   Discard collection tube with eluate and place in a new 2 mL collection tube	____

   *Verify addition of ethanol to concentrate
   **If additional drying time is required, centrifuge for 1 additional minute
   Comments:

9. DNA Elution

   Pipet 200 µL of elution solution into the center of the column	____
   Incubate column for 5 minutes at room temperature	____
   Centrifuge for 1 minute at 6500 x g*	____

   *Template DNA remains in the eluate

   Comments:

10. Prepare PCR Ingredients
    Instructions for Primer resuspension can be found at primers.htm

    Add 25 µL of NEB Master Mix to a 2 ml PCR tube	____
    Add 2.5 µL of forward primer 10 µl	____
    Add 2.5 µL of reverse primer 10 µl	____
    Add 20 µL of template DNA	____

    Comments:

    Forward Primer:
    Reverse Primer:
    

11. Run PCR

    Place the PCR tube in the machine and set the cycle as follows (repeat x30)

    Step	Temperature	Time
    1	94 C	30 seconds
    2	55 C	30 seconds
    3	72 C	1 minute

    Comments:

12. PCR Product Purification

    Add 25 µL of Buffer PB to 5 µL of PCR reaction and mix*	____
    Put QIAquick column in 2 mL collection tube	____
    Apply Sample DNA to the column	____
    Centrifuge Column in tube for 60 seconds at 17,900 x g	____
    Discard the flow-through and place the column back in the 2 mL tube	____

    *If orange or violet add 10 µL 3M sodium acetate

    Comments:

13. PCR Product Wash

    Wash Column with 0.75 mL of Buffer PE	____
    Centrifuge for 60 seconds at 17,900 x g	____
    Discard the flow-through and place the column back in the 2 mL tube	____
    Centrifuge for 60 seconds at 17,900 x g	____
    Place column in a clean 1.5 mL microcentrifuge tube	____

    **Use Multichannel pipette

    Comments:

14. PCR Product Elution

    Add 50 µL of Buffer EB to the center of the QIAquick membrane	____
    Centrifuge column for 60 seconds at 17,900 x g	____

    Comments:

15. Running Agarose Gels
    Instructions for running agarose gels for analysis of PCR products can be found at Agarose Gel Instructions