Demystifying PCR: fostering scientific literacy through hands-on PCR education

Now we get to add the DNA to the Reaction Mix!

The reaction mix contains the enzyme Taq which is the thermostable DNA polymerase. This is the enzyme which can add nucleotide to the opposite strand during PCR. 

It also contains the primers. Primers are usually added in separately for specific purposes. The primers are what binds to the specific DNA sequence of your target (gene or promoter or whatever region), and allows the Taq to bind and start copy the template DNA strand. Further details are available in the Lab Notes slides. 

For the purposes of this Starter Kit, we have prepared the mixes containing each of the primers, the Taq, the nucleotides and buffer ahead of time. In addition, primers remain more stable in a reaction mix than in sterile double distilled water. This decreases the chance of any cross-contamination. If you want to make your own mixes up, the appendices will have all the methods to make these mixes for expanding the kits.  

Still there will be pipetting, and the pipetting resources we will send in the kit should come in handy. We will publish the resources in the pipetting section. Remember to mix the sample smoothly that you are pipetting. Also wear a mask and work in a PCR hood, if you can. Working in a PCR hood or culture hood can help prevent contamination from aerosols generated during mixing. 

Materials: 

A. the small PCR reaction tubes labelled: 

   CS-cow, sus1-cow, sus2-cow, ctrl-cow and CS-pig, sus1-pig, sus2-pig, ctrl-pig

B. Required Components produced in earlier steps: 

Extracted DNA or control in the 4 "QE" tubes <-- These are your test samples that will be added to the PCR tubes.  

C. positive controls and negative controls in 4 small PCR tubes with blue writing with " * " sign:  

   +ve cow, +ve pig, -ve pig, -ve cow. <--- these tubes mailed with the kit are complete and ready with mix, primer, template. They will serve as positive and negative controls having DNA in them, which has already been tested in our lab. 

Methods: 

Adding in the DNA to the tubes: 

This is simply adding 2 uL of the extractions to the tiny PCR tubes. We have written all the steps out and provided a pictogram as well. We will PCR all the DNA samples using two different sets of primers - one set for cow and the other for pig. 

for the cow PCR: 

As in the pic above: 

 - add 2 uL of the sample QE-CS tube into the PCR tube labelled CS - cow

 - add 2 uL of the sample QE-sus1 tube to the PCR tube labelled sus1 - cow

 - add 2 uL of the sample QE-sus2 tube to the PCR tube labelled sus2 - cow

 - add 2 uL of the sample QE-ctrl tube to the PCR tube labelled ctrl - cow

for the pig PCR: 

as in the pic above: 

 - add 2 uL of the sample QE-CS tube to the PCR tube labelled CS - pig

 - add 2 uL of the sample QE-sus1 tube to the PCR tube labelled sus1 - pig

 - add 2 uL of the sample QE-sus2 tube to the PCR tube labelled sus2 - pig

 - add 2 uL of the sample QE-ctrl tube to the PCR tube labelled ctrl - pig

While it might seem like a lot of pipetting if you're new to it, at least you are not loading a 384 well PCR plate.... yet!

Now you are ready to Cycle and perform the polymerase chain reaction. Next step is just to program the PCR machine appropriately. See the next protocol note please!