Gel purification of large plasmids for PCR or sequencing, HELP! - (Mar/17/2008 )
Hey all,
I need some assistance please!
I am investigating resistance genes on large (50kb up to possibly 170kb) plasmids in [E. coli.
I have extracted them fine and separated them on agarose gels to 'plasmid profile' my isolates.
The isolate have 1-3 plasmids each and I would like to perform PCR to determine which plasmid contains which gene.
The usual gel purification kits (like spin columns, etc) only go up to 25kb (if your lucky!). I have been researching electroelution/dialysis methods, but have never used or know anyone who has used them.
Does anyone have any suggestions or an alternative method that I could use to confirm which resistance genes is carried on which plasmid?
I am a visiting international student, so I don't have lots of time to play with.
Thanks in advance!
dear boswell
I am using Mazi GeBAflex tube from GEBA in order to elute proteins from acrylamid gel
these tubs works perfectly as expected and i also read that they are compatible with DNA extraction from gel
after electroelution i dialysis with PBS
bye
I need some assistance please!
I am investigating resistance genes on large (50kb up to possibly 170kb) plasmids in [E. coli.
I have extracted them fine and separated them on agarose gels to 'plasmid profile' my isolates.
The isolate have 1-3 plasmids each and I would like to perform PCR to determine which plasmid contains which gene.
The usual gel purification kits (like spin columns, etc) only go up to 25kb (if your lucky!). I have been researching electroelution/dialysis methods, but have never used or know anyone who has used them.
Does anyone have any suggestions or an alternative method that I could use to confirm which resistance genes is carried on which plasmid?
I am a visiting international student, so I don't have lots of time to play with.
Thanks in advance!
[size="2"]large plasmids for PCR in Gel purification is simple, efficient, well established rapid process 50kb - 200kb. Using Gebaflex-tube in electro elution mode generate 70 to 90% recovery. used for the past 3 years+ with consistant effectivness.