Agarose gel electrophoreses for PCR products ? - (Jan/04/2011 )
I m having microsatellite PCR products of size ranging from 116 bp to 318 bp, what would be the concentration of agarose for gel electophoresis for visualization of these products. Should i perform simple PAGE or urea PAGE for it ?? ( right now i m out of chemicals needed for PAGE thats why i wanted to go for gel electrophoresis for initial screening) I once tried 4 % agarose but not able to make it, there were too many bubbles & froth when i heated it in a microwave, i tried intermittent heating also but that is also not working ?
Pls help me out.
2% agarose should work fine, though it will not give you much separation of similar size products. If you need to determine the differences between similar sizes use acrylamide gels.
Thanx bob1.......I just need to know whether PCR amplification is occurring or not , i will be using urea PAGE for differentiation.
Thanx a lot
If you only need to separate the 116 bp from the 318 bp, there is absolutely no need for PAGE. You can use a 1.5-2% agarose gel with the TAE buffer or even better the SB buffer. There was recently a thread on this forum about separating 31 bp from 36 bp on an agarose gel using optimized low concentration buffers.
Hi again everyone,
I have to run Urea PAGE now for my PCR products, my product size range is 116 bp to 300 bp, for what time should i pre run & run the gel, what is the criteria for deciding the run time & pre run time ? & also i want to stain the gel by silver staining so what conc. of silver nitrate should i use. My assembly is a little big one , its biorad sequi gen & requires a lot of buffer ........