low melt agarose - (Dec/09/2010 )
I have 2 qsts.
Is it ok to run MY RESTRICTION DIGEST MIX IN low melt agarose overnight at room temp as I am running pout of time? Will it melt if I run it at 20 V o/n. any chances of bands gettng diffused? I cut the Lentiviral vector (12728bp) with EcoR1 and am expecting the bands at 10453 bp (which I need to cut from gel to later insert the gene of interest) and 1704 bp. I want the bands well separated (cut and uncut part).
other qst is that I am using 1% LMP agarose as I can handle it but have not tried lower concentrations. Will it be sufficient enough to separate these big size bands?
I am new in cloning studies.
1st question, it is certainly okay to run a gel overnight at low voltage. A fragment that size will not diffuse laterally much.
Personally I have a feeling that 1% agarose will not give good resolution between 12.7kb from 10.5kb. it would be better to aim for a good complete digest by giving EcoRI sufficient time to cut all the DNA.