PCR for Genetic Screening 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. (Sample A, Sample B, Sample C, Positive control(+), or Negative control (-)) Place the tubes on ice.
2. Each group prepares a master mix. Label an 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.




for each PCR Reaction	Components	6X Master Mix (you prepare this)
31.8μl	ddH2O	190.8μl
10.0μl	5X Taq PCR Buffer	60.0μl
6.0μl	MgCl2	18.0μl
1.0μl	dNTPs (2.5mM each)	6.0μl
1.0μl	Forward Primer (100pmol/μl)	6.0μl
1.0μl	Reverse Primer (100pmol/μl)	6.0μl
0.2μl	Taq Polymerase	1.2μl
use 48μl per reaction		288.0μl Total

3. Pipet gently to mix the master mix and store on ice.
4. Add 48.0 ul of the master mix to each sample tube and the control tubes. Pipette so that the mix is on the bottom of the tube. Keep the tubes on ice.
5. Centrifuge the DNA tubes and then add 2.0 ul of the appropriate template DNA to each reaction tube. Pipette reaction mix so the DNA template is mixed with the Master Mix in the reaction tube. Keep the tubes on ice.
6. If needed, tap the sample tubes to bring the liquid down to the bottom. Return tubes to ice
7. Add a drop of mineral oil to the top of each sample, then spin all PCR reactions briefly in the centrifuge. Return the tubes to ice.
8. Place tubes firmlyh into the thermal cycler to ensure good thermal contact and begin cycling according to the following parameters:



Initial Denaturation 96°C 2 min

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

Prepare for Electrophoresis

1. Prepare a 2% agarose gel by adding 2 g of agarose to 100 ml of
   1x Sodium Borate buffer in a flask. Microwave the flask for about 2 minutes, until agarose is 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.
2. Prepare the gel tray by bringing up the dams on the ends of the tray and carefully tightening the screws snuggly, but not too tight. If the gel tray does not have attached dams, then firmly tape the ends to create dams.
3. Place the 6-well comb into the slots at the top of the gel.
4. 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.).
5. Add about 325-350 mls of 1x sodium borate running buffer to the electrophoresis chamber.
6. 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. If the level of the running buffer in the electrophoresis chamber does not cover the gel, then add more so that the gel is covered.
7. 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.
8. Fill in the blanks in the table below with the label of the DNA template in the tube that you plan to load in each lane of the gel. with the wells at the top of the gel, Lane 1 is the well on the left side.

Sample Preparation and Loading

1. Label a 0.5 ml tube with the name of the template DNA (A, B, C, + and -).
2. Add 2μl FOTO/Vision^TM loading dye to the tube.
3. When the reactions have finished cycling, use a P20 pipette to remove 10μl of PCR sample to the labelled tube with 2μl FOTO/Vision^TM. and pipette up and down several times to mix. Be sure that the pipette tip is underneath the top layer of oil when you draw your DNA!
4. Load 6μl of the DNA Marker and 10μl of each sample with dye into the appropriate wells of the gel.
5. 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.
6. Turn on the power supply and set it at 220 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 20 to 25 minutes.
7. After the gel has run, remove it from the gel box. Drain off as much buffer as possible. Place the gel on the UV light box to visualize the DNA and to photograph it.

PCR Notes

Forward Primer (Tm = 59°C) (100pmol/μl)

 5’ CGCCAGGGTTTTCCCAGTCACGAC-OH 3’

Reverse Primer (Tm = 50°C) (100pmol/μl)

 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.