My palsmid is around 9000 bp in pSectag2-MycHis-IRES-EGFP vector with ampicillin resistance and use DH-alpha E.coli.. It is highcopy plasmid. I tried both large and small volume of culture medium. When i do mini prep to confirm the plasmid using 2 ml of culture from large culture (200ml to 500ml), i get quite good amount of plasmid for example 0.8 to 1.0 ug/ul eluted with 10 to 20 ul TE). But after conformation, when I do Max-prep the plasmid yield is very low for example 0.2 ug/ul to 0.5 ug/ul eluted with 100 ul TE 300-500ml culture media. One year ago i faced the same problem. i used several reagent from different company but did not get any solution but finally it was solved by some of my personal tricks but now all of my tricks are failed and i am getting crazy about this.
How did you overcome finally for that problem? can you give me some suggestion?
Maxiprep usually gave low yeild specially with high molecular weighr plasmid. I think try to use midiprep or adjust bacterial culture to get the best culture time to obtain high yield
You could try to eluate your plasmid using hot water, at 65-70°C. It help the release of the plasmid from the column.
I recently did one maxi prep with pSectag2 vector and 2000 bp insert. I managed to get a concentration at 2200 ng/ul in 200 ul elution. Which Kit are you using? we use qiagen and Zymo. My mini prep con is almost 800 ng/ul in 50 ul elution volume. heat up your elution buffer at 60 for 20 min and incubate at least 2 mins after adding this elution buffer onto the membrane provided with the kit.
If it with the same culture, then you have a problem with your column, always column has a capacity of mg of DNA, so please check your coulmn efficiency. you almost missed 30 times of your DNA concentration, ether it didn't solved the the solvent buffer or didn't eluted by DW or TE buffer.
you may need to optimize each step separately to know the cause or try to change your method of preparation.
this paper may helps you
Article Protocol: A rapid and economical procedure for purification ...
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