ISSR protocols:

Primer sequences used in the Wolfe Lab for the three case studies presented in the workshop overview section (obtained from publications, or designed in the Wolfe Lab-- most are based on the UBC set):

7 (CT)8-RG 901 (GT)6-YRTERRY (GTG)4-RC
814 (CT)8-TG 902 (GT)6-AYMAO (CTC)4-RC
843 (CT)8-RA AW3 (GT)6-RGMANNY (CAC)4-RC
844 (CT)8-RC M1 CAA-(GA)5GOOFY (GT)7-YG
898 (CA)6-RY M2 GGGC-(GA)8BECKY (CA)7-YC

Standard Reaction conditions (25 µL volume)

DNA 0.5 無
primer 0.5 無 (20 to 50 然)
Taq buffer 2.5 無 (10X)
dNTPs 4.0 無 (1.25 mM)
ddH2O 15.9 無
Taq 0.1 無 (5 U/無)
MgCl2 1.5 無 (50 mM)

Thermocycler program
(we use a Stratagene Robocycler)

1.5 min at 94°C (melt); 35X 40s @ 94°C, 45s @ 44° or 45° C, 1.5 min @ 72°C; 45s @ 94°C, 45s @ 44°C, 5 min @ 72°C; 6°C soak

Miscellaneous notes

We usually run our reactions with a 20 to 50 µM primer concentration.

I purchase my primers through Genosys (usually about $12 per primer with our institutional discount). University of British Columbia offers a kit for about $250 that includes 100 primers (I think that's the correct number). I've heard mixed reports about the quality of the primers offered and that's why I opted to use previously published/modified sequences or primers I designed instead of going with the kit. However, one should keep in mind that these short oligos tend to go bad in a shorter amount of time than longer primers one might use for DNA sequencing projects. That could account for the rumors about the UBC primers. The UBC set does offer a lot of primers to try and would be a good initial investment for screening many primers at the beginning of a study. Individual primers could then be purchased separately.

I haven't tried these reaction conditions on any other machine, so you may have to play with conditions for your DNAs. We usually run a temperature gradient with each new primer and then adjust [Taq] and [MgCl2] for the next rounds of optimization.

We've had some mixed results with our clean-up protocols (Sephaglas-based procedures). We usually have best results with just diluting the raw DNA extract and standardizing the template. However, in several studies the raw DNA extract wouldn't amplify consistently and clean-up of the DNA was required. We use the Elu-Quik protocol for cleaning template DNAs.

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Last updated January 17, 2000.