4 replies to this topic

[Dr vet]

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.

Thanx

[bob1]

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.

[Dr vet]

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  

[BioMiha]

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.

[Dr vet]

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 ........