I have done Mini and maxi preps with macherey nagel kits and had no problems.
Tried Sigma GenElute Endotoxin free plasmid Midiprep kit for the first time in DH5alpha cells.
Starting culture 50ml, followed the exact protocol (eluted in 1ml as indicated)
Result - OD: 6ng/ul A260/280: 1.54 A260/230: 0.74
Any suggestions of what would have went wrong?
I should use this plasmid for transfecting HEK293 Tcells.
Well, did you save various fractions? If you did, electrophoreses those to find out at which step you had the problem. I would also like to confirm that selection antibiotic(s) was not bad and added into the culture medium in correct concentration.
I have not saved various fractions.
I added 50ul of Ampicillin into 50ml LB culture. The Ampicillin that I used was in 4C and not so old (took from -20C four days back).
Perhaps in this case you want to repeat the extraction one more time. If it was Ampicillin, please double the concentration i.e., 100 ug per mL. Just to be on the safe side, do confirm that non transformed DH5 are not growing in ampicillin medium. Save fractions and see what you get. With all that data to back up your claim, you can ask the company to replace the kit if reagents in the kit were bad or something like this.
Thank you Syed.
I repeated the experiment changing some parameters that yielded high pure plasmid. 260/280: 1.88 260/230: 2.11 OD: 107.9ng/ul
1. Lowered my culture to 20ml.
2. Was careful when separating the aqueous phase from the endotoxin phase.
3. After the final DNA wash, spun (5000g for 2min) the column with the empty collection tube again (so it removed any residual ethanol in the column).
4. Preheated the endotoxin free water to 65C and added 500ul of that water into the column and let in sit for 10 min before I spun down for elution.
Increasing the culture volume to 30ml might yield higher concentrations of DNA.
try PureLink or GeneJet. very reliable kits, high plasmid DNA yield
the latest product launch, released this week - PureLink Fast kits (Invitrogen). ultraclean plasmid DNA in 30 min: up to 0.4 mg (midi) and 1.5 mg (maxi scale)
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