This page provides complete PCR instructions for use with Carolina Haplotyping Kit.
Hair sheath DNA isolation
- Mix 200ul of proteinase K with 200ul of Chelex solution in a 0.5ml eppendorf tube. The Chelex has tiny beads. Make sure they are well suspended in solution before using. You can close the tube and flick it with a finger to help suspend the beads. Keep this mixture on ice or in refrigerator until ready to use. Prepare this mixture the same day you want to use it.
- Each person submitting a sample should tweeze 3-5 hairs with good sheaths or large roots. Eyebrow hairs work well. See the Carolina manual for examples of what to look for. Cut off the hair just above the sheath and discard the portion without a sheath. Drop the hair sheath sections into a 0.5ml eppendorf tube. Label the tube with the participant's initials. Add 100ul of the proteinase K/Chelex mixture to each tube. Make sure the sheaths are submerged in the mixture and not stuck to the side of the tube.
- Incubate the tubes for 10 minutes in a 50C water bath.
- Remove the tubes from the water bath and vortex the samples or manually flick each tube for 15 seconds to help dislodge cells from the hair sheaths.
- Incubate the tubes in a boiling water bath for 8 minutes.
- Remove the tubes and allow to return to room temperature, cooling for 2 minutes.
- Vortex or flick the tubes for 15 seconds.
- Spin the tubes in a centrifuge for 30 seconds or allow to sit for 5 minutes to allow any debris to settle.
- Transfer 50ul of the supernatant to a new 0.5ml eppendorf tube and discard the old tube. Label this new tube with the participants initials and a unique number. (Ex: HB1, PS2, ED3, etc.) Keep this on ice or stored in the freezer until ready to use. It can be stored for several weeks.
- Put ice in an ice bucket. Retrieve PCR bead tubes (one for each sample) and number the lids. The numbers should correspond to the numbers you gave to the DNA isolation samples. Retrieve the DNA samples and the pink human mitochondrial DNA primer mix tube. Keep everything on ice. Some items, like the primer mix, may be frozen solid. It is okay to warm these in your hands or pockets until they are thawed, but avoid over-heating. Set tubes back on ice as soon as they are in a liquid state.
- Transfer 22.5ul of the primer mix into each PCR bead tube. Use a new tip every time. Do not touch the pipette tip to the PCR bead. (It will dissolve immediately and stick all over the pipette tip.) It's easiest to expel the liquid onto the side of the tube and allow it to sink down to the bead either by gravity or by closing the tube and tapping or shaking the liquid down to the bottom.
- Transfer 2.5ul of DNA into each tube. Make sure the numbers match. (Ex: DNA from HB1 should go into the PCR tube labeled 1, DNA from PS2 should go into PCR tube 2, etc.) Use a clean tip for each transfer and use the same precautions as in the previous step - do not allow the tip to touch the PCR bead.
- Close all the tubes and make sure all the liquid has reached the PCR beads and dissolved them. Keep everything on ice as often as possible.
- Open the tubes and place one drop of mineral oil into each tube. Do not allow the mineral oil tube or dropper to touch the PCR tubes at all. Hold the mineral oil tube above the PCR tubes and let the drop of mineral oil fall into each tube.
- Close the PCR tubes. They are now ready to go on the thermocycler. It is best to put them on the thermocycler right away, but if you need to wait, keep them in the freezer until you are ready to start the reaction.
Running the PCR Reaction
- Have the PCR tubes ready and on ice.
- Start the thermocycler and allow it to ramp up to the first denaturing temperature of the progam (94C). Set the tubes into the thermocycler immediately, or just before it reaches the denaturing temp.
- Let the reaction run through all the cycles. Once it is complete, take the tubes out of the thermocycler and immediately put them on ice or store them in the freezer.
Pouring a 2% Agarose Gel
- You will need 50ml of unused 1x TBE buffer to pour the gel. You will also need another 500ml of 1x TBE buffer to run the gel, but this amount can be used from previous gels, and can contain Carolina Blu stain in it. If you need to mix 1x TBE buffer, use the following procedures:
- Mix 30ml of 20x TBE buffer with 570ml of distilled water.
- Label the container with it's contents and the date you mixed it. TBE will last at room temperature for a couple years.
- To the 500mL used to run the gel, you will need to add 24 drops of Carolina Blu stain. Swirl to mix thoroughly.
- Prepare the casting tray by taping off the ends. Make sure the tape is water-tight so that nothing will leak out of the casting tray. You can test it by running water from the sink into the tray, but be sure to dump out all the water and blot the casting tray as dry as possible before proceeding.
- Use the gram scale to weigh 1g of agarose and put it in a clear, microwave-safe container - preferably one with a narrow neck like an Erlenmeyer flask. Pour in 50ml of 1x TBE buffer. Swirl the flask to mix the contents together. The agarose will not dissolve, but you want it to be somewhat suspended - at least not in a large chunk - before moving further.
- Put the flask in the microwave and set the microwave for 2 minutes. You will need to watch the flask carefully as it is being microwaved. As soon as it hits boiling point and you see bubbles forming, open the microwave and swirl the flask to mix the contents. (You will want an insulated glove - a glass container will be very hot.) There will likely still be some undissolved agarose in the flask. Keep repeating this process, microwaving it, stopping it and swirling it whenever it begins to boil, until you can no longer see any undissolved agarose.
- Now you want to cool the flask slightly. You can do this by holding the outside of the container under cool running water from the sink while swirling the flask. Do not allow any water to get into the flask and do not stop swirling it. You do not want the agarose to solidify right now. If you notice the agarose is forming chunks, put it back in the microwave to melt it and try again.
- Once the flask is cool enough that you could touch it with a bare hand, but no clumps have formed, add 2 drops of Carolina Blu gel stain and swirl the flask to mix thoroughly. Immediately pour the gel into the casting try. Do this in one smooth pour to avoid bubbles forming in the gel. If you do form any bubbles, you will need to rake them to the end of the gel using the comb.
- Set the comb into the gel right away. Make sure that the tips of the comb penetrate the gel surface, but do not poke all the way through to the bottom of the gel. Put the entire casting tray with comb into the refrigerator until the gel is completely solidified. Use the gel within one day of pouring it, the fresher the better.
Running the Gel
- Remove the casting tray with the solidified gel from the refrigerator. Set the casting tray into the gel box and carefully remove the comb. If any wells tear while you remove the comb, you will not be able to use those wells.
- Pour in enough 1x TBE buffer to completely cover the gel. (If you have not done so already, be sure to add the 24 drops of Carolina Blu stain to the TBE before pouring it into the gel box.) You do not want a large volume of buffer above the gel surface - just enough to cover it.
- Put ice in an ice bucket and set your PCR reaction tubes in the ice. Retrieve the pBR322/BstNl tube and keep it on ice as well. It may be frozen solid. You can warm the tube in your hands until it thaws, but avoid over-heating.
- Transfer 10ul of pBR322/BstNl into one lane of the gel. This will be your ladder. When pipetting into the gel, keep the tip just at the top of the well. Do not try to stick the tip down into the well. When you press down on the pipette's plunger, the liquid should flow down into the well on its own.
- Transfer 5ul of each PCR product into its own well in the gel. The PCR tubes will have a solid layer of mineral oil covering the red PCR product. You want to poke the pipette tip through the mineral oil and into the PCR product before trying to pull any liquid up into the pipette tip. If the mineral oil is not solid, you may need to keep it on ice longer. You want to be able to poke through it as a solid layer, rather than having oil stuck to your pipette tip.
- Make sure the power supply is hooked up such that the wells are closer to the negative end and the DNA can run through the gel towards the positive end.
- Turn on the power supply to 130V. You should see bubbles forming along the wires within the first few minutes.
- Run the gel until the loading dye from the pBR322/BstNl lane has moved at least 2 inches. Be sure to stop the gel before the loading dye runs off the end of the gel and into the buffer tank.
Staining and Destaining the Gel (optional)
- Set the gel and casting tray into the staining tray. Pour enough final stain to just cover the gel. Let it sit for 20 minutes.
- Over the sink, remove the gel and casting tray from the staining tray and pour distilled water over the gel to rinse it. You may pour the final stain from the staining tray back into the reagent bottle as long as you do not get any distilled water in it.
- Set the rinsed gel and casting tray into the destaining tray. Pour in enough distilled water to cover the gel. Let this sit for 15 minutes.
- Rinse the gel in distilled water over the sink again. Dump out the water from the destaining tray, set the gel and casting tray back into the destaining tray, and refill it with fresh distilled water. Allow this to soak for at least 30 minutes and up to overnight.