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Help! Plasmid disappearing after mini-prep.


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#1 Trana

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Posted 07 October 2009 - 02:24 PM

Hi, I have recently ligated a 500bp insert in to the pET28a vector (NcoI, EcoRI). Transformation seems to be successful, as there are around 20-30 normal sized colonies on the plate. I grew a series of cultures to screen for my insert, and the Kanamycin I used was freshly prepared. The cultures grew just fine, and after around 5-6 hours, the cultures were turbid. So, I did the mini-preps, and then tested analyzed using UV-Vis. A260 indicated I have around 150ng/uL, and A260/280 is 1.7. However, when I run these samples in any quantity on an agarose gel, NOTHING shows up, not even a smear to indicate degradation. Any suggestions??

Thanks!

#2 phage434

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Posted 07 October 2009 - 03:33 PM

Does your marker lane show up?

#3 Trana

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Posted 08 October 2009 - 08:15 AM

Marker lane shows up, as does a digested control vector that I run

#4 hanming86

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Posted 13 October 2009 - 04:27 AM

maybe

1. contamination, no plasmid . might wanna smell ur culture and see if it got the E.coli scent
2. maybe something wrong with ur plasmid prep. A260 could be from RNA as well, but somehow i feel it as quite unlikely as RNAse is normally included in most prep


How was ur minipreps done? this is quite hard to determine. the gel part seems fine. control was there. no dye floating out i assume (etoh contamination ) when loading?
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