Separating a 2750 base DNA band from a 2600 base DNA band - (Oct/17/2008 )
Hi everyone
After doing a digest of my shuttle plasmid, I need to separate and purify a 2750 base DNA fragment from a 2600 base DNA fragment via agarose gel purification. Since the sizes are so close (only 150 bases apart) it is impossible to distinguish between the two on the gel (I used 0.7% gel). Does anyone have any suggestions on a better approach?
thanks
Lisa
After doing a digest of my shuttle plasmid, I need to separate and purify a 2750 base DNA fragment from a 2600 base DNA fragment via agarose gel purification. Since the sizes are so close (only 150 bases apart) it is impossible to distinguish between the two on the gel (I used 0.7% gel). Does anyone have any suggestions on a better approach?
thanks
Lisa
1. Could try double digest i hope u're not using double digest at the moment..
2. 0.5% gel for the win, run the the gel for a long long time.
After doing a digest of my shuttle plasmid, I need to separate and purify a 2750 base DNA fragment from a 2600 base DNA fragment via agarose gel purification. Since the sizes are so close (only 150 bases apart) it is impossible to distinguish between the two on the gel (I used 0.7% gel). Does anyone have any suggestions on a better approach?
thanks
Lisa
1. Could try double digest i hope u're not using double digest at the moment..
2. 0.5% gel for the win, run the the gel for a long long time.
thanks
There may be (likely is) a restriction site on the vector which is not on the insert. Cut with this enzyme as well, which will result in two shorter vector bands.
thanks
another simpler approach is to try to run the sample on higher % gels.
run the sample on 1.5-2% gels and run for a longer time (say 30-40 min or more).
you can easily distinguish dna of 100-200 bases apart.
run the sample on 1.5-2% gels and run for a longer time (say 30-40 min or more).
you can easily distinguish dna of 100-200 bases apart.
thank you
Use a high purify agarose like metaphor with a concentration of 2-3% and run it at a lower voltage than normal.
To prepare a metaphor gel it must be prepare in cold buffer and let it "hydrate" for ten min with continous stirrnig, then when melting it must be doing carefully because it burns easily. After polymerize let it at 4-8C until use for a better separation of bands.
To prepare a metaphor gel it must be prepare in cold buffer and let it "hydrate" for ten min with continous stirrnig, then when melting it must be doing carefully because it burns easily. After polymerize let it at 4-8C until use for a better separation of bands.
thank you