K-12 Programs

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PCR Field Trip
Protocol

Set-up the PCR Reactions

1. Label a 0.5 ml, thin-walled PCR tube with your initials and the number of the DNA template that you are using. (A, B, or C, positive or negative control) Place the tube on ice.

2. Each group prepares a master mix. Label a clear 0.5 ml tube with MM. Prepare enough mix for the number of reactions plus one. (Ex. 3 samples + 1 negative control + 1 positive control = 6x) Keep all of the components and the mix on ice.

PCR Reaction	Components	6X Master Mix
1.0ml	Template DNA  (100ng/ul)	N/A
5.0ml	10X Taq PCR Buffer	30.0ml
1.0ml	Forward Primer	6.0ml
1.0ml	Reverse Primer	6.0ml
1.0ml	dNTPs	6.0ml
40.8ml	ddH2O	244.8ml
0.2ml	Taq Polymerase	1.2ml
50.00ml		use 49ml/reaction

4. Pipet gently to mix the master mix and store on ice.

5. Add 49.0 ul of the master mix to each sample tube and the negative control tube. Keep the tubes on ice.

6. Add 1.5 ul of the appropriate template DNA to each reaction tube. 

7. Use the tube with the remaining master mix as a balance tube. Microfuge the sample tubes to bring the liquid down to the bottom.

8. Add 1 drop of mineral oil to each reaction tube. Return the tube to ice.

9. Program the thermocycler. Initial Denaturation 96°C 2 min

5 cycles:
Denature 94°C 45 sec 
Anneal 50°C 30 sec 
Extend 72°C 1 min

10. Place tubes firmly into the thermocycler to ensure good thermal contact and begin cycling.

1. Prepare a 2% agarose gel by adding 2 g of agarose to 100 ml of 0.5x TBE. Microwave for about 2 minutes, until completely dissolved. Allowing agarose to hydrate in the buffer before the mixture is microwaved may make it easier to fully dissolve the agarose. The agarose is cooled to about 55°C before adding ethidium bromide and pouring the gel.

NOTE: Ethidium Bromide (Et Br) is a mutagen; it enters the cell, intercalates into the DNA and can mutate it. Always wear gloves, lab coat and safety glasses when using EtBr.

2. Prepare the gel tray by bringing up the dams on the ends of the tray and carefully tightening the teflon screws snuggly, but not too tight. Or firmly tape the ends.

3. Place the 6-well comb into the slots at the top of the gel.

4. Add 5 ul EtBr to 100 mls of 55°C agarose and swirl gently (don’t form bubbles) to mix.

5. Pour the agarose into the middle of the tray until it is about half way up the teeth of the comb and has filled the tray to the corners. Do not disturb while the agarose is solidifying (about 20 mins.).

6. Add about 325-350 mls of 0.5x TBE to the electrophoresis chamber.

7. After the gel has set, lower the dams, or carefully remove the tape. Hold the tray on the high side, with the comb closest to the black electrode, and slip it into the electrophoresis chamber on top of the platform. The dams, if present, should hang down over the ends of the platform.

8. Rock the comb very gently, back and forth in the gel, to allow a little buffer into the well around the teeth. Gently remove the comb and rinse it with ddH2O. Remember, the gel contains EtBr.

10. Fill out the table below, writing the number of the DNA template in the lane that you plan to load. By convention, the lane of the gel closest to you is lane 1.

1. Label a 0.5 ml tube appropriately for the template DNA (A, B, C, positive and negative controls).

2. Add 2ul of 6x loading dye to the tube.

3. After the reactions have finished cycling, use a P20 pipette to remove 10 ml of sample from the reaction tube. Depress the plunger in the air and go through the oil layer before releasing the pressure to draw up the sample from the bottom of the tube.

4. With a Kimwipe quickly and carefully wipe the oil from the outside of the pipette tip.

5. Add the 10 ul of PCR sample to the labeled tube with 2 ul of 6 x loading dye and pipette up and down several times to mix.

6. Load 10 ul of the 100 bp DNA Marker in lane 4 of the gel.

7. Load 10 ul of each sample with dye into the appropriate wells of the gel.

8. Place the lid on the gel box; connect the electrodes to the power supply. Make sure that the black wire goes into the black plug and the red into red.

9. Turn on the power supply and set it at 130 V. Check the milliamps to make sure that the current is running. Bubbles at the electrodes also indicate that the current is running. The gel will run for 45 minutes.

10. After the gel has run, remove it from the gel box. Drain off as much buffer as possible. Remember that the gel and buffer both contain EtBr. If your gloves are wet from the buffer or handling the gel, change them before touching anything else in the lab. Place the gel on the UV light box to visualize the DNA and to photograph it.

10x PCR Buffer Forward Primer Tm = 59°C 100pmol

100 mM Tris-HCl (pH 9.0 @ 25°C) 5’CGCCAGGGTTTTCCCAGTCACGAC-OH 3’

500 mM KCl

1% Triton - X 100 Reverse Primer Tm = 50°C 100pmol

25 mM MgCl2 5’TCACACAGGAAACAGCTATGAC-OH 3’

dNTP’s are 2.5 mM of each dATP, dTTP, dGTP, dCTP for a total of 10mM dNTP’s. The final concentration of all four nucleotides in the reaction is 200uM.

Taq DNA Polymerase is a thermal stable enzyme that was isolated from an organism (Thermus aquaticus) found living in geyser pools in Yellowstone Park. Taq polymerase, like most polymerases, requires a primer, which is a small piece of DNA ending in a 3’ -OH group, bound to the single-stranded template DNA. The enzyme sits down on this small stretch of double-stranded DNA and begins to travel down the single-stranded template adding complimentary nucleotides as it reads the template.

Mineral oil is added to the reaction to prevent evaporation at the higher temperatures, which would change the concentrations of the components in the reaction and inhibit the polymerase.

• Background
• Genetic Screening Scenario
• Information for Teachers
• Set-up
• Biotechnology Field Trips Home Page

Contact: Barbara Bielec, 608-273-9737