POLYMERASE CHAIN REACTION

Protocol I (50ml reaction volume):

This protocol is used with commercially purchased Taq DNA Polymerase/dNTP/2X buffer premixes (e.g., PanVera).

Prepare a Primer Premix by adding 1000pmol of each primer to a microcentrifuge tube. Bring the volume up to 100m l with deionized water. Keep cold. For storage, the 10X primer mix can be stored at -20° C. The working concentration of this premix is 100pmol of each primer.

Thaw PCR tube containing the Taq DNA Polymerase/dNTP/2X buffer premix on ice. To each tube add 25m l of Taq DNA Polymerase/dNTP/2X buffer, 10ml of the Primer Premix, and 1-2m l of template DNA. Bring the final volume to 50ml with dH₂O

Cycle this mixture through an appropriate program on the thermal-cycler.

Protocol II (50ml reaction volume):

This protocol is used with commercially available Taq with 10X buffer

Thaw the tubes containing the Taq DNA Polymerase, Taq DNA Polymerase 10X buffer, and dNTP on ice. To each reaction tube add 10ml of the Primer Premix, 1-2m l of template DNA, 5ml 10X buffer, and dNTPs to a final concentration of 2.5mM. Bring the volume to 50ml with dH₂O

Protocol III (100ml reaction volume):

This protocol is used to prepare the 10X buffer for use with commercially available Taq DNA Polymerase.

Reagent	Stock Concentration	Working Concentration
MOPS, pH 7.75	20mM	0.2mM
MgCl2	30mM	3mM
KCL	500mM	50mM
dNTPs	2mM each	200m M each

Once these stock reagents are made, filter sterilize the MOPS and dNTPs, and autoclave the MgCl₂ and KCl.

To make 1000m l of 10X buffer/primer premix (enough for ten reactions), add the following:

100m l MOPS

100m l MgCl₂

100m l KCl

100m l dNTPs

X m l Appropriate primers (1000pmol of each primer)

dH₂O to one ml

Use 100ml of this buffer for each reaction. Best results are achieved using 0.5mg to 0.8mg genomic DNA. Some changes may need to be made when applying this to other types of DNA. Add 0.5 units Taq per reaction.

Cycle this mixture through the appropriate program on the thermal-cycler.

This same procedure is used for symmetric or asymmetric amplifications, depending on what the amplification is for (dot-blot, sequence etc.). The only difference between the symmetric and asymmetric will be the ratio of the amplification primers.

Purification of amplified DNA:

Mineral oil (if used during the PCR) can be separated from the reaction mix by pipetting the entire mix onto a piece of Parafilm. By "rolling’ this mix around on the Parafilm, the oil from the mix will be separated from the aqueous PCR mix. The aqueous DNA containing solution can then be removed and the DNA isolated without oil contaminants.

Pipette the amplified DNA into a fresh microcentrifuge tube. To this add 1 volume of 3M NH₄Oac and 2 volumes of 85-95% ethanol. Vortex briefly and centrifuge at maximum speed for 15 minutes. Discard the supernatant and wash the pellet with cold 70% ethanol. Discard the wash (re-centrifuge if the pellet becomes dislodged during the wash) and place the tube top down on a paper towel to allow all traces of ethanol to drain. While holding the tube top-side down, gentle aspiration can be used to eliminate all traces of ethanol. Redissolve the pellet in an appropriate volume of water or TE.

Analysis of amplfied DNA by gel electorphoresis:

Pour an agarose gel that will allow visualization of the amplified DNA (e.g., 1% agarose gel).

Pipet 10% of the reaction volume into a microcentrifuge tube. To this add water to bring the volume to at least 10m l and 2m l of loading dye. Alternatively, the use of a microcentrifuge tube can be eliminated by mixing the three on a piece of Parafilm.

Load each sample into an individual well. To one well, add an appropriate DNA marker.

After the run, visualize the amplified DNA using UV excitement of ethidium bromide.

Jackson's Laboratory | Protocols | Reagents