make your own gotaq - (Jan/05/2010 )
I am working up a prac for a visting high school student group that are coming to our uni to learn about mol biol etc.
We are trying to do it with as little manipulation on their part as possible. One major bottleneck with our undergraduate program is loading gel with students taking for ever to add loading dye to their samples and then loading them on the gel so we want to elimante this with the high school students as we only have them for an hour. I have got my thermalcycling protocl down to under 20mins and gel running times down to 5 mins so i am anticipating this as being my major hold up.
I have used Gotaq in my research career but cost per reaction is restrictive for this excercise.
Has anybody had a go at making their own PCR additives that allow them to go straight from thermalcycler to gel loading.
Any feed back would be greatly appreciated.
I have not tried it, but I believe the dye in the Sigma ready-load pcr mix is cresol red. You should be able to add Ficoll or sucrose to make the pcr mix dense without much of a problem. Have you thought about the Invitrogen E-gels, which need no loading buffer? Expensive, but with lots of wells and fast running.
I made a 40% sucrose solution with 0.25% xylene cyanol and 0.25% orange G in PCR water (basically loading buffer) and used this as a 5X stock in the PCR master mix and it worked great. No impact on the PCR, sunk into the wells fine and good colour.
Would also like to say a big thanks to the people from Promega who contacted me just after i tried this, offering me some free products for use with the high school groups.