Genotyping PCR reactions

This PCR protocol is for genotyping microsatellite loci. Reactions are typically set up for a 96 well plate for genotyping on the 377. Primers can be multiplexed (multiple primers in a given reaction).

Reagents you will need

5x or 10x buffer

Mg free buffer aliquots are kept in general freezer. Use the buffer that goes with the Taq. For GoTaq, use 5x buffer without the added green dye. The buffer contains KCl, Tris (pH 9) and Triton X (a detergent which keeps the polymerase happy). The Triton X makes it pretty bubbly. Bubbles can be removed by a quick centrifuge spin.

Magnesium chloride, 25 mM

Some people add MgCl2 directly to the PCR buffer. However, it can also be added separately to the PCR mix. This enables optimization of the Magnesium concentration. Tubes are typically kept on the door of the small freezer.

dNTP mixture, 10 mM total; 2.5 mM each

Thaw 100 mM stock solutions of dATP, dCTP, dGTP and dTTP (in freezer at end of sequencer bench). Add 10 ul of each plus 360 ul of water to an O ring tube. This makes a total of 400 ul and dilutes each 1-> 40 making them 2.5 mM.

There is an optimal Mg concentration to maximize the efficiency of the Taq. This occurs at about 1 mM Mg. dNTPs bind Mg in solutions. If there are more dNTPs than free Mg, then the polymerase has no Mg to activate it and the reaction will not work. With 2 mM Mg adding dNTPs to 0.8 mM so that there is 1.2 mM free Mg works great.

Primers

Typical working concentrations of primers are 20 uM. This can be made from 200 uM stocks by diluting 1-> 10 (i.e. 20 ul up to 200 ul).

Primers and DNA are always diluted with TE/10 (that is 0.1x TE = tris / EDTA)

Taq polymerase

Stock solutions of this enzyme are kept in the -20 freezer (top shelf). Taq seems to get inactivated by multiple warming cycles (bringing out to room temperature). So, keep your working tube to a few weeks supply and try and use a freezer box or put it on ice.

PCR working reagents are kept in the freezer (-20C) when not in use. This prevents buffer and primer degredation, and Taq inactivation.

Setting up a PCR reaction

For making up master mix, mix everything except DNA. Either 20 or even 10 ul reactions can be run since only 2 ul is loaded on the gel. Make up about 10% extra since there are always some pipetting losses, e.g. to do 96 PCR reactions, make up enough for 100??:

When using 5x buffer

Reagent	For 1 rxn	For 100 rxn
5X buffer	4.0 ul	400 ul
25 mM Mg	1.6 ul	160 ul
dNTP	1.6	160
Forward primer	0.2	20
Reverse primer	0.2	20
Taq polymerase	0.15	15.0
Water	11.25	1125
Volume w/o DNA	19 ul	1900 ul
Volume w/ DNA	20 ul

When using 10x buffer:

Reagent	For 1 rxn	For 100 rxn
10X buffer	2.0 ul	200 ul
25 mM Mg	1.6 ul	160 ul
dNTP	1.6	160
Forward primer	0.2	20
Reverse primer	0.2	20
Taq polymerase	0.15	15.0
Water	13.25	1325
Volume w/o DNA	19 ul	1900 ul
Volume w/ DNA	20 ul

Use a clean pipette tip for each reagent. Once all master mix reagents have been added, vortex briefly to mix and spin down for 1 min in centrifuge. Aliquot 24 ul into each tube of a 8- or 12- tube strip (can use same tip for this).

Add DNA, mixing each well. se a clean tip for each DNA well so no cross contamination. Put lids on tubes. Label each strip on ends w/ date at least. Spin down.

Final reaction concentrations.

Other hints and options:

You can use 10 ul reactions as only 2 ul of each reaction gets loaded on the gel.

For multiplexing, the HEX (yellow) labeled primers are not as bright at the FAM (blue) or TET (green) labels. To compensate, you may want to add 2x or 3x as much of the HEX primer. The total amount of primer should still be 0.8 mM or less in a multiplex reaction.

For 96 rxns, you can use an 8-strip tube to aliquot the master mix with a multi-channel pipettor.

For primers that are designed for 60 C (standard), the typical cycling conditions are

95 deg C for 2 min

95 deg C for 30 sec
54 deg C for 30 sec
72 deg C for 30 sec
Cycle 28 times