1
Standard Paq polymerase PCR
The Antonovics Lab uses this protocol often in tandem with “5% Chelex DNA extraction” to
extract and amplify Microbotryum DNA
1. Fill a couple of buckets with ice.
2. Take desired DNA samples out to thaw and place them on ice.
3. Place necessary amount of tubes and plates on ice.
a. You will make one eppendorf tube of a ‘master mix’ that is then divided amongst
each DNA sample.
b. You will need one 8-well strip for large sample sizes, this way you can spread out
your master mix after it is made and use the multichannel pipette to fill your plate.
c. For small numbers of samples, use 8-well strips, for larger amounts use PCR
plates (these can be cut for less than 96 samples)
d. Let them sit on the ice for a few minutes to cool before adding reagents.
4. Make your master mix by following the recipe below.
a. This example is for 16 samples of DNA.
b. Add 0.5 µl extra in your multiplication for any sample size between 1 and 40, 1.0
µl from 41-96, and 1.5 µl from 97-192 to account for pipetting error or mistakes.
You may use larger extra volumes until your pipetting becomes accurate enough.
c. Paq buffer is very bubbly, avoid excess pipetting to reduce bubbling.
d. The Paq polymerase should not be taken out of the freezer until you are ready to
add it LAST. It does not freeze, so just take it out, add it, and put it back in the
freezer immediately.
e. Be sure to mix your other reagents once they have thawed and before adding by
flicking with your finger a few times and then spinning them down.
i. DO NOT mix Paq polymerase. It will simply get bubbly and make
pipetting accurately nearly impossible
f. Be sure to mix your master mix once all ingredients are added in the same
manner. If you master mix gets bubbly because you flicked it too much, try to
spin it down or pray that your pipetting still works out.
Reagent
ddH2O
10x Paq Buffer
dNTPs
Forward Primer
Reverse Primer
Paq Polymerase
Amount per
rxn (µl)
6.4
1.0
0.1
0.2
0.2
0.1
+ 2µl of
Multiply
Total Amount to put
by (n+0.5) in master mix (µl)
16.5
105.6
16.5
16.5
16.5
1.65
16.5
3.3
16.5
3.3
16.5
1.65
DNA template per well
5. Distribute 8 µl of your PCR master mix into each well.
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a. Do this before adding DNA samples so that you can use one tip.
6. Add 2 µl of your DNA samples to each well, a new tip for each so as not to contaminate.
a. Using the P2 pipette is recommended to have greater thumb control
7. Seal 8-well strips with 8-well caps using black sealing pen or seal 96-well plates with
adhesive films.
a. Be sure to fully seal across the plate, if seal is not full, evaporation will occur.
b. If you send your plates out to be sequenced, use 8-well caps on plates to prevent
evaporation.
8. If drops remain on the sides of your tubes, spin them down until no bubbles remain and
all of the mixture is in the bottom of the wells.
9. Place on thermocycler with the following program.
a. 95 ºC for 2 minutes
b. 30 cycles of:
i. 95º C for 20 seconds
ii. 55º C for 20 seconds
iii. 72º C for 30 seconds
c. 72º C for 5 minutes
d. 4º C hold
10. Afterward, store in –20º C freezer for storage or use immediately for analysis