I am trying to separate and analyze macromolecules (DNA) using horizontal gel electrophoresis. Protocol involves 1X TAE buffer, 10X denaturing buffer, 1X running buffer, 1% agarose gel, and 2X denaturing loading buffer. Once the buffers are prepared and gel is cast, upon loading the samples into the gel, I have to place the gel in the 2-8 deg C fridge for about 16 hours. Occasionally the power supply fails, or worse, the fridge fails! I'm desperate for alternatives. I don't understand why a) the run time is so long (something to do with ladder I believe) and b) why the gel must be run in such low temperatures? Any gels I ran during university were done at room temperature. Any/all insights are appreciated!
Nahed Hussien
Thank you for those suggestions! My team will give them a try.We are currently running at 40V, so 80V sounds like a promising fix.
cheers!
16 hour runs used to be normal for Southern blots. using 20cmx 20cm gels and were run at room temperature ( UK about 20c). The separation was between 37kb and about 100bp. The reason for keeping cool was to minimise thermal broadening by diffusion of the smaller bands when they were close together. The larger bands are not noticeably affected by warmer running.
Smaller gels would need lower voltages but I agree with Nahed Hussien
that dna is very stable and faster running and higher temperature is fine so long as the buffer is fresh- Badges
- Science topic
- Similar topics
- Gels