I got a yield of greater than 5 microgram/ul of my plasmid preparation but the 260/280 ratio was low i.e. 1.38 using Nanodrop. I want to use the plasmid preparation for transformation experiment in future. Is there any procedure that I can follow to decontaminate the present preparation from protein ?
If you only need it for transformation, there's no need for further purification. BTW, how did you measure concentration? Also by nanodrop, or did you check an aliquot by electrophoresis?
Then check 1 uL of both the plasmid and a 1:100 dilution by electrophoresis. If it really has a conc. of 5 ug/uL it will be visible in both cases. What's your 260/230 ratio?
Didn't noted down the 260:230 ratio. One more thing, the solution gave precipitate after keeping at 4 degree celsius overnight !!
the plasmid that you have now should work for transformation after removal of the precipitate by centrifugation. But if you need greater purity for other applications, you can try the following steps.
You can first dilute the plasmid with TE buffer and re-check concentration using nanodrop. If the precipitate that was formed after overnight storage at 4 C still does not dissolve, you can remove it by centrifugation. Collect the supernatant and do a phenol/ choloroform extraction followed by chloroform extraction step. collect aqueous phase, add 1/10th to 1/5th volume of Na-acetate (3M). Mix well. Add 0.7 times the volume of isopropanol. Incubate at room temperature for 5-10 min. Centrifuge at max speed for 5 min. wash pellet twice with 70% ethanol. Air dry pellet and redissolve in TE. Measure concentration. dilute plasmid to ~ 0.5-1.0ug/ul and re-check on nanodrop. confirm plasmid integrity on gel.
hope this helps.
all the best.
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