I used a CHEF MAPPER XA system and I used the following parameters: 5-40s , 19h , 6V/cm, at 14°C. What should I do to get a good bands? what is my problem?
There are a few factors can affect your gel resolution, such as the way the samples processed, the thickness of the gel, how the gel is stained, and temperature of the run the actual running condition etc.
If your samples are in liquid form, make sure do not let it overflow. IF you samples are in plugs, make sure there is no air bubbles in the plug
Always set up the pump circulation at least 30 min before your run, make sure the chamber temperature reaches the setting temperature, as well, leave the gel in the chamble for 15-20 minutes pre chill before run the gel
If you stain your gel after the run, please allow the stain at 4 degrees for 30 mins then de stain for 5-10 minutes before take the image, if you had dye in your running buffer, please always remember to de stain your gel for 3-5 minutes using fresh buffer without dye before photography the gel.
If your samples was digested before run (guessed) you may need to dialyze to get rid of excess of the sault in the sample before lead
Consider load the gel only using half amount of the samples
If the above conditions are still not able to give you significant improvement, you may consider to lower the temperature to 7degrees and lower the high end of the pulse, using 5-30s
Good luck
Lingli
Hi Samira,
As you were already told, a number of factors can affect the resolution of PFGE. You may find useful to have a look at the troubleshooting section in Pulsenet.
Best of luck
http://pulsenetinternational.org/protocols/troubleshooting/
I found the type of stain you use to stain your gel is crucial. I was using redgel and I never got clear bands. Now i am using midori green advance (5 micro-liter to stain 100 ml of 0.7% agarose gel) and together with all the other factors for (example the time and the voltage which should be adjusted to the size of your sample) it worked perfectly.
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