Band Shift Optimisation - (Apr/06/2001 )
I mī starting a work with mitochondial DNA of Fishes, I need the secuences of a pair of universal primers for molecular sistematic purposes.I have search and found many, so i want to know about real performances. Thanks in advance.
Re: your problems with bad resolution in gel shift assays- I have a few suggestions:
1. Try different PAGel concentrations- my GRAs work best with an 8% gel
2. Experiment with different Acrylamide:bis ratios. The usual 29:1 ratio may not be the best because the protein(s) may be large and may require a more porous gel to run well. I use a 75:1 acryl:bis ratio which I dissolve in enough MQ water to make a final concentration of 30% e.g 75g acrylamide + 1 g bis in 100 ml water will be a 76% solution so you could dissolve them in a larger volume to get a final conc. of 30%
3. Try using 0.5X TBE or even 1X TAE as running buffer (of course the gel should also be cast in the same concentration of buffer)
4. Good quality fresh acrylamide solutions make a big difference! Make a fresh stock and keep it in the dark at 4 degress C after filtering through a 0.45 micron filter. Don't use acrylamide solutions which are more than 3 months old.
5. Experiment with different binding buffers. Do a literature search for buffers that other investigators use for gel shifts with the same protein, if it's a known protein, and try out diferent ones. One of them at least should work well under your experimental conditions.
6. Check what the half life of the protein-DNA interaction in the gel is. You could be running the gel for too long and the protein(s) could simply just fall off the DNA by the time you stop the run, so you'll end up with a smear. Try running it in the cold room- after a 15 minute or so pre-run- for different time periods.
Usually one sees this sort of problem if the labeling reaction is not complete. You should label the oligo to completion by adding cold nucleotide for some more time (say another 10 minutes), if u are using the the klenow reaction. The fragment is different sizes differing by one nuceotide and hence u see a smear.Good luck.Momin