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No plasmid after a maxi prep mystery


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4 replies to this topic

#1 LostintheLab

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Posted 12 April 2012 - 02:19 AM

Here's a conundrum for all you molecular biology experts.

We have 7 constructs (all the same vector backbone) from the same company, we have managed to get good amounts of plasmid yield from 4 of them. The remaining 3 constructs will transform fine, but when we grow them up and then maxi prep- we get no DNA from the maxi prep.

Any suggestions, answers, help?
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#2 Trof

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Posted 13 April 2012 - 01:53 PM

Are the other three constructs larger or/and contain repetitive sequences or toxic product?

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#3 LostintheLab

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Posted 19 April 2012 - 07:26 AM

Hi Trof,

In answer to your questions
The constructs should all be the same size within a few base pairs, we don't know the exact sequence- they are shRNA constructs, so although they should silence a gene, they shouldn't be toxic to cells. If it were a toxic product I would anticipate a lack of bacterial cell pellet after growing up, but we don't see that ( this part I may be wrong on). The pellets are large- there just doesn't seem to be any DNA when we maxi prep.
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#4 phage434

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Posted 19 April 2012 - 11:41 AM

Make sure you keep antibiotic selection. If ampicillin, then switch to carbenicillin as the antibiotic. Losing plasmid selection gives a large pellet and no plasmid DNA.

#5 LostintheLab

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Posted 23 April 2012 - 02:59 AM

Thanks Phage- might try that. Its a little frustrating as all the other constructs maxi prepped fine, but then didn't work in our experimentPosted Image
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