5
$\begingroup$

I'm teaching a class how to do PCR & gel electrophoresis soon and would like it if you could check through my basic step by step instructions - it's a while since I've done one. Is there anything there that is wrong, missing, or should be excluded in the following?

A) Collect all ingredients from the refrigerator and keep cool (on ice). Leave the TAQ in the freezer until required.

B) Make master mix by adding all ingredients (ddH20, MgCl2, PCR Buffer, F Primer, R Primer, dNTP) (excl. TAQ)

.......

W) Take the gel to the UV camera and take a picture. Large DNA molecules move through the gel slowest so will be nearest to the wells.

(The students will use primers they designed to sex 6 DNA samples from 3 species of birds, males should produce 1 band & females 2 bands in the gel)

for the full version see the answer below

Improve this question
$\endgroup$
9
  • 1
    $\begingroup$ for one thing you have to add a stain to the gel to see it on the UV. ethidium bromide requires gloves and is toxic. GelGreen/GelRed is expensive, but is a lot safer. also remember to keep the mastermix frozen at -20C until use - aliquot it because you can't refreeze it more than ~6 times or so - it degrades. $\endgroup$
    – shigeta
    Nov 11, 2013 at 18:22
  • $\begingroup$ Thanks @shigeta useful tip of freezing and aliquot in the mm. I forgot the loading dye is a prepared mix of loading dye and gelred $\endgroup$
    – rg255
    Nov 11, 2013 at 19:15
  • 1
    $\begingroup$ @GriffinEvo Exactly, and then do PCR on that sample alongside all the others and run it on the gel. $\endgroup$
    – A. Kennard
    Nov 11, 2013 at 19:56
  • 1
    $\begingroup$ here's a good PCR troubleshooting guide.. neb.com/tools-and-resources/troubleshooting-guides/… $\endgroup$
    – rg255
    Nov 11, 2013 at 20:34
  • 2
    $\begingroup$ Looks good to me, with the comments others have shared. I think the most important part of preparing a lab like this is not only make sure you get decent results (although with master mixes and the like these days that's usually pretty straightforward), but to know exactly WHY you're doing each step, and to be able to explain that clearly to the students so they understand. Anybody can follow a protocol, but it takes a special person to connect the "how" with the "why" and explain it all in a unified story. Good luck with your lab! $\endgroup$
    – MattDMo
    Nov 12, 2013 at 5:55

1 Answer 1

Reset to default
4
$\begingroup$

I've run a test following this protocol to the letter...

PCR step by step:

  1. Collect all ingredients (excluding TAQ) from the refrigerator and keep cool (on ice). Leave the TAQ in the freezer until required.
  2. Make master mix by adding all ingredients (excluding TAQ) using fresh pipette tips for each ingredient and using the volume specified in the PCR reaction mix recipe.
  3. Vortex & lightly spin down on a centrifuge.
  4. Add the required amount of TAQ to master mix (insert in to the liquid) and mix using the pipette to draw some liquid up and down.
  5. Add master mixes to labeled PCR tubes in the required volumes as specified by the protocol.
  6. Add DNA to PCR tubes (using fresh pipette tips for each DNA sample) again using the volume specified in the protocol. Label your tubes carefully (and on both the lid and tube).
  7. Lightly spin down the PCR tubes on a centrifuge.
  8. Run PCR in the thermocycler according to your protocol.
  9. Collect samples from thermocycler and place in fridge until needed.

Gel Electrophoresis step by step:

  1. Make 1xTAE (enough to make the agarose gel and to place in the electrophoresis apparatus).
  2. Make an agarose gel (about 5 mm thick) by melting agarose and 1xTAE in the microwave; allow the liquid to cool a little before adding it to the mold.
  3. Once the gel is cool, place it in the electrophoresis apparatus, cover it with 1xTAE (just covering the gel) and then remove the comb.
  4. Collect the PCR products from the fridge/thermocycler.
  5. Make spots of loading dye on parafilm. The loading dye is premixed with GelRed so your samples show under UV light.
  6. Add one sample to a spot, mix it by drawing the liquid in and out of the pipette tip, and then carefully add to a well in the agarose gel. Take care not to puncture the gel or spill any of the liquid out of its well. Discard the pipette tip.
  7. Repeat the previous step for each sample and for a negative control (master mix and milli-q water instead of DNA). Use a fresh tip for every sample.
  8. Load PCR Ladder, also mixed with loading dye, in to a well.
  9. Connect the electrophoresis apparatus to a power supply, with negative electrode (black) nearest to the wells containing the samples.
  10. Set the power to run 80-120 volts.
  11. Wait until about a finger-width gap appears between the blue and purple bands (approximately 20-30 minutes).
  12. Disconnect the power and collect the gel carefully from the liquid.
  13. Take the gel to the UV camera and take a picture. Large DNA molecules move through the gel slowest so will be nearest to the wells.

And got a perfect result...

enter image description here

The order is Control, female, male, female, male, female, male, ladder. Males have one band, females have two. The final pair didn't show too well on the picture but were clear enough on the screen in the camera room. Thanks for the tips everyone! Sorry the picture is low quality.

Feel free to recreate these guidelines but remember to credit the Biology Stackexchange Community!

Improve this answer
$\endgroup$
1
  • $\begingroup$ now that's dedication! Thanks for sharing this! $\endgroup$
    – shigeta
    Nov 13, 2013 at 18:13

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .