In past I have seen that gDNA runs well on 0,8 gel (home made gel agarose) but with precast gel I haven't had the possibility of choosing the right percent of agarose.
Hi Vincenzo, why shouldn't it work on a 1,2% gel?! You have to run the gel a little bit longer or increase the voltage. Cheers, Nadine
because genomic DNA is larger in size (eg:E.coli-4.6MB) you may have to use low percentage agarose gel such as 0.8% for the separation of larger molecular DNA. 1.2% percent is normall used to separate small sized (say 300-500bp) PCR fragments.
Thank you so much for your answer, Nadine. I am going to use for the first time a precast 1,2% agarose gel to appreciate integrity of genomic DNA. My doubts are about the possibility of a wrong running of the high molecular weight DNA with a higher percent of agarose.
Ananda, i know that 0,8 % is a better agarose concentration for genomic DNA but I have to use a precast gel. The only parameters that I can change are voltage and time. I use precast gel from Invitrogen and their composition with SIBR Safe is only 1,2%. Do you think that I will see a wrong DNA migration although setting longer running time and higher voltage? please, help me
Out of my curiosity, why you have to use this company made gel? what is SIBR safe? is there any thing special with the precast gel(no EtBR staining required or some other chemical is there to help your analysis?). If nothing is useful, I would simply pour my gel.
All I know is, the resolution of molecule is inversely proportional to the %. Say for example what happen if I run a 200,300,400,500nt mixture of DNA in 0.5% gel, we wont see the separation at all (no matter how less time you run...right?). The same apply if you have larger DNA sized above the threshold for higher percentage gels(porosity/percentage of agarose) in which it has to pass through (I mean to say we cannot force a 20kDa molecule into 2kDa cut off filter...you know.....).
I think you are seeing your DNA on the wells after loading? please let me know what is the case. I can learn either.
@Ananda: It's called "SYBR safe": http://www.lifetechnologies.com/de/de/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/dna-stains/sybr-safe.html
Cheers, Nadine
Oh I heard of these EtBr supplements...thanks Nadine.
I routinely use 1% home made agarose gel for the detection as well as quantification of genomic DNA.
- Badges
- Science method