You will need your DNA extractions (1), an empty PCR tube (2), an empty 1.5 mL microcentrifuge tube, the primer mix for this project (3), Firepol master mix (4), and PCR grade water (5).
The first step is to calculate how much PCR reaction mix you require for your samples.
PCR reaction mix is the combination of Firepol master mix, primer mix and PCR grade water you will add to each PCR tube before adding your DNA extraction.
For each sample, you will need:
- 4 µL of Firepol master mix
- 12 µL of PCR grade water
- 2 µL of primer mix
- 2 µL of DNA template
You also need a negative control.
A negative control is a PCR tube of PCR reaction mix that you do not add DNA to. This is used to check your PCR reaction mix is not contaminated.
If you are doing multiple reactions using the same primers, you should first make a batch PCR mix containing all of the shared reagents, rather than creating each PCR from its individual components. This means less pipetting, and you will use far fewer pipette tips.
To calculate how much of each reagents you will need:
[number of DNA extractions] + [negative control] + 10%
For example, if you have 9 samples from your DNA extractions:
9 DNA extractions + 1 negative control + 10% = 11 repeats of PCR reagents
- 11 x 4 µL = 44 µL of Firepol master mix
- 11 x 12 µL = 132 µL of PCR grade water
- 11 x 2 µL = 22 µL of primer mix
In this example, you would use the 20-200 µL adjustable pipette to transfer the 44 µL of Firepol master mix, 132 µL of PCR grade water and 22 µL of primer mix into a 1.5 mL microcentrifuge tube. Make sure to use a fresh pipette tip each time.
Close the lid of the 1.5 mL microcentrifuge tube and invert several times to ensure thorough mixing of your PCR reaction mix.
If you are using a batch PCR reaction mix, once you have produced the batch PCR mix as above, set the 2-20 µL adjustable pipette to 18 µL and transfer 18 µL of PCR reaction mix into the required number of PCR tubes.
If you are not using a batch PCR mix (e.g. only doing one or two samples), use a 2-20 µL adjustable pipette to add each item (4 µL of Firepol master mix, 12 µL of PCR grade water and 2 µL of primer mix) individually to the PCR tube.
Use a permanent marker to label the PCR tubes with your sample names. Label the negative control too so you know not to add DNA to this PCR tube.
Now add the DNA extraction. Set your micropipette to 2 μL.
Using a fresh pipette tip, transfer 2 μL of your DNA extraction into the correspondingly labelled PCR tube containing PCR reaction mix. Then discard your tip.
Make sure to keep your DNA extraction upright and pipette from the surface of the liquid.
The DNA extractions contain PCR inhibitors that will prevent your PCR from being successful if the liquid is mixed.
When you have pipetted the DNA extraction into the PCR tube, close the lid and invert the tube several times to ensure the DNA is mixed into the PCR reaction mix.
Tap the PCR tube firmly on a hard surface to collect the liquid at the bottom and to ensure there are no air bubbles in the liquid.
Place your PCR tubes in the thermocycler block.
Set up the thermocycler with the following PCR program:
CHD1F/CHD1R primers
- 4 mins at 94°C (or 15 mins at 94°C if using 5x HOT FIREPol® Blend Master Mix Ready to Load)
- 8 cycles made of 3 steps:
- 30 sec at 94°C
- 45 sec at 57-50°C (touchdown)
- 45 sec at 72°C
- 27 cycles made of 3 steps
- 30 sec at 94°C
- 45 sec at 50°C
- 45 sec at 72°C
- 5 mins at 72°C
- ∞ at 15°C
(For help setting up a touchdown PCR on your Bento Lab visit the PCR Thermocycler User Manual.)
If you need help operating the Bento Lab thermocycler, check the manual. You can use the PCR preset (1), then modify (2) the program to the required settings (3) before running the program (4).
The program will run for ca 2 hours. When it is finished, you can keep the result in the freezer, or use it right away for gel electrophoresis.
