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Question about plasmid midiprep


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#1 jamestoon

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Posted 26 April 2011 - 09:20 AM

I am using Promega's PureYield™ Plasmid Midiprep System.

1. The protocol mentioned that I should start with inoculating my bacteria in 1-10ml LB media and incubate them for 8 hours before diluting them 1:500 and re-incubate in larger culture volume for 12-16 hours.
What if I already incubate my starter culture for 24 hour before the dilution and re-incubation steps? Can I just dilute it and not incubate it? Or should I assume that I have not incubated them for so long and just continue to dilute and re-incubate?

2. Before plasmid isolation, we need to add isopropanol to Endotoxin Removal Wash and 95% ethanol to Column Wash.
What if I don't have isopropanol? Can I add 95% ethanol to both Endotoxin Removal Wash and Column Wash? Will this affect the yield/concentration of plasmid?

3. The protocol also mentioned that we should use only a centrifuge with a swinging bucket rotor.
What if I don't have a centrifuge with a swinging bucket rotor? Can I use normal centrifuge without the swinging bucket rotor?

Thanks :)

#2 pDNA

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Posted 26 April 2011 - 10:04 AM

first of all sticking to the protocol is not a bad idea at all :)

1) if you have grown your starter culture for 24 hours the cells have been hanging around a long time in the worn out medium, acidifying it, dying, losing recombinant plasmids ...and so on ...so i would not assume that they are fit and possibly something happend to your construct ...but who knows. If you want to you can dilute them and re-incubate them for another 12-16 hours (make sure to use selective pressure by adding antibiotics to your freshly diluted culture and grow them). I personally would do it the way they have stated in the protocol ...but you can give your approach a try.

2) If you don't have isopropanol you can skip that step ...possibly leading to more impurities in your preperation but as long as you don't use your plasmid for transfection of eukaryotic cells it should be okay.

3) Using the static rotor instead of the swing-out makes no real difference for the cells ...but you should take more care when you handle the pellet since it is attached to the wall and not to the bottom of the conical tube. But dont worry about that ...just take care not to loose it.

Good luck!

Regards,
p

I am using Promega's PureYield™ Plasmid Midiprep System.

1. The protocol mentioned that I should start with inoculating my bacteria in 1-10ml LB media and incubate them for 8 hours before diluting them 1:500 and re-incubate in larger culture volume for 12-16 hours.
What if I already incubate my starter culture for 24 hour before the dilution and re-incubation steps? Can I just dilute it and not incubate it? Or should I assume that I have not incubated them for so long and just continue to dilute and re-incubate?

2. Before plasmid isolation, we need to add isopropanol to Endotoxin Removal Wash and 95% ethanol to Column Wash.
What if I don't have isopropanol? Can I add 95% ethanol to both Endotoxin Removal Wash and Column Wash? Will this affect the yield/concentration of plasmid?

3. The protocol also mentioned that we should use only a centrifuge with a swinging bucket rotor.
What if I don't have a centrifuge with a swinging bucket rotor? Can I use normal centrifuge without the swinging bucket rotor?

Thanks :)






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