I'm trying to make recombinant protein, but after digesting my inserts, and purification via NucleoSpin extract kit, I always end up with poor plasmid concentration and very low 260/230 ratio, even though i tried to consider the possible mistakes! and when i continue the transformation with the same DNA, i can see no bands at all on the gel, although the bacterias are growing.
Does anybody had similar experience or have some ideas to help me overcome this problem?
The critical point to get low amount is during loading. Try to add loading solution 2 times, some protein may be traped in filter
regarding poor plasmid concentration issue, you can elute the plasmid in lower volume in elution buffer. u can also pass the mix after adding buffer 3 twice through the column. regarding 260/230 ration try to increase the number of thanol washes. if the problem still persists, you can repurify plasmid using phenol:chlorofoam:isoamyl alcohol treatment 24:25:1 followed by isopropanol wash n ethanol wash n elution in evn small volume. you will get proper 260/230 ration as well as good plasmid yield.
as suggested earlier, it is possible that ethanol is still there in your plasmid solution so the plasmid actually does not enter the well of the gel n floats instead in the buffer. heat the plasmid at 37 degrees for 5 mins so that ethanol gets evaporated n then load the plasmid solution. also do not incubate with solution 2 for longer time. it lowers 260/230 ratio.
thank you sansrity , i guess the only thing i didn't try is to heat it up..
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