Opsin PCR reactions
This PCR protocol is used for opsin gene amplification. It may be different from some of the genotyping protocols in three regards. First, the PCR buffer includes the Mg. This reduces the number of things to add (or forget) in the master mix. Second, the dNTP concentration is 4 time higher (10 mM each dNTP rather than 10 mM total dNTP). This is compensated for by the fact that 1/4 as much is added. Third, the primer concentrations are 10 uM (some of the genotypers use 20 uM). This is because the opsin stock primers are 100 uM (rather than 200 uM). None of this makes a difference as the concentrations in the final PCR reactions are the same.
Reagents you will need
10x buffer containing 20 mM MgCl2
Mg free buffer aliquots are kept in general freezer (3rd from door)
This 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.For each 1 ml of Mg free buffer, add 20 ul of 1M MgCl2. You may need to measure the buffer volume with a pipettor.
You can also add MgCl2 separately.
Solution 10X concentration Working 1X concentration KCl 500 mM 50 mM Tris HCl pH 9 100 mM 10 mM Triton X 1% 0.1 % MgCl2 20 mM 2 mM
dNTP mixture, 10 mM each
Thaw 100 mM stock solutions of dATP, dCTP, dGTP and dTTP (in freezer at end of sequencer bench). Add 30 ul of each plus 180 ul of water to an O ring tube. This makes a total of 300 ul and dilutes each 1-> 10 making them 10 mM.
N.B. This is four times more concentrated than some people in the lab use, however we use 1/4 as much is the PCR reaction so it all works out.
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 I typically add dNTPs to 0.8 mM so that there is 1.2 mM free Mg which works great.
Primers
Typical working concentrations of primers are 10 uM. This can be made from 100 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
GoTaq polymerase is kept in the -20C freezer. Other Taq stock solutions are kept in the -80 C freezer. We have shown that storage at -80 or -20 C is equivalent. However, some of the Taq gets inactivated by multiple warming cycles (bringing out to room temperature). So, keep Taq in a cold box when you remove it from the freezer.
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
Typical recipes scale directly with final reaction volume, eg. 25 or 50 ul
Reagent Final conc 5X GoTaq buffer 5 ul 10 ul 1x 25 mM MgCl2 2 ul 4 ul 2 mM dNTP (10 mM each = 40 mM total) 0.5 1 10 mM total Forward primer (10 uM) 1 2 0.4 uM Reverse primer (10 uM) 1 2 0.4 uM GoTaq polymerase (5U/ul) 0.1 0.2 0.5-1 U sd water 14.4 29.8 DNA (30-50 ng) 1 1 Total volume 25 ul 50 ul
For making up master mix, mix everything except DNA. Make up about 10% extra since there are always some pipetting losses, e.g. to do 10 PCR reactions, make up enough for 11:
Reagent For 1 rxn For 11 rxn 5X buffer 5 ul 55 ul 25 mM MgCl2 2 ul 22 ul dNTP 0.5 5.5 Forward primer 1 11 Reverse primer 1 11 Taq polymerase 0.1 1.1 Water 14.4 158.4 Volume w/o DNA 24 ul 264 ul Volume w/ DNA 25 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.
PCR machine
Everyone keeps their own programs on the PCR machine. Use a program which first denatures DNA, then runs 30-35 cycles of PCR and then cools to 4 C. This is a method which links other programs. The denature step is 95 C for 1 min or so. Check the PCR program to change annealing temperature or times. Typical PCR conditions are: 95 C 20 s / 53 C 30 s / 72 C 1 min repeated for 35 cycles.