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Miniprep / plasmid question


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#1 assembler01

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Posted 26 May 2009 - 07:04 AM

I normally grow overnight cultures for my minipreps. We're looking for mutagenized plasmid (by quickchange type protocol) and I've been having trouble with this particular mutant. This is more of a general question however. Suppose I picked some colonies from a LBKan20ug/mL plate (with a pEGFP based mutant protein EGFP fusion) and did an overnight culture-Kan20. Some grew a little and I did a miniprep and got nothing by spectrophotometer, but I'll be checking them by EtOH precip/gel later. Some apparently didn't grow but I threw them back in the incubator overnight again. They did all grow by the next day but it was a weekend so I threw them in the lab and they sat still at RT for 2 nights. Is it possible to get plasmid out of these guys assuming they had it? Would they have lost the plasmid by now because kanamycin probably ran out and we weren't selecting for it during the 2 nights?
How long have any of you grown cultures (or left at room temp/4degC) of transformed bacteria that you picked from colonies and still got plasmid out?

#2 Vini

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Posted 26 May 2009 - 07:22 AM

I normally grow overnight cultures for my minipreps. We're looking for mutagenized plasmid (by quickchange type protocol) and I've been having trouble with this particular mutant. This is more of a general question however. Suppose I picked some colonies from a LBKan20ug/mL plate (with a pEGFP based mutant protein EGFP fusion) and did an overnight culture-Kan20. Some grew a little and I did a miniprep and got nothing by spectrophotometer, but I'll be checking them by EtOH precip/gel later. Some apparently didn't grow but I threw them back in the incubator overnight again. They did all grow by the next day but it was a weekend so I threw them in the lab and they sat still at RT for 2 nights. Is it possible to get plasmid out of these guys assuming they had it? Would they have lost the plasmid by now because kanamycin probably ran out and we weren't selecting for it during the 2 nights?
How long have any of you grown cultures (or left at room temp/4degC) of transformed bacteria that you picked from colonies and still got plasmid out?



I have never left my cultures at RT for that long. Though, left them at 4C once for an entire day. there were few cultures from which I cud extract DNA but there were also some which had gone bad. That is, i did not get any DNA. Anyhow, i dont like keeping the cultures even at 4C for couple of days.

#3 microgirl

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Posted 26 May 2009 - 08:27 AM

Because they took so long to grow up I would think there was something wrong with your constructs in the first place. If you were desperate to resurrect them you could try streaking them out on a kan plate for reisolation and trying to grow them up again.

#4 eldon

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Posted 27 May 2009 - 04:28 PM

Because they took so long to grow up I would think there was something wrong with your constructs in the first place. If you were desperate to resurrect them you could try streaking them out on a kan plate for reisolation and trying to grow them up again.


sounds reasonable...shouldn't take that long...no growth overnight is a bad sign and leaving them longer likely means what does grow is not what you want.

if they did grow well o/n....it's a different story...i have prepped plasmid from o/n cultures that sat at room temp for 3 days..yield was a little low, but i still had more than enough for sequencing etc.....i have prepped plasmid from o/n cultures that were kept at 4C for an entire week without a problem either...it's like a stock plate at 4C (they last about a month if kept cold and sealed properly to avoid dessication of your colonies.

#5 Functional Screens

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Posted 27 May 2009 - 06:25 PM

16-20 hours should give you a "cloudy" culture, if not, sometimes it's because bacteria DO NOT like what you put in. The gene product might be expressed in the bacteria and makes them "unhappy". I will suggest you to try 30C and 25C (then 20-24 hours are needed) to see whether you can get more to grow. Also, you can try SOB, SOC, or even TB. Especially TB might give you 10 times more bugs....
My two cents, ALWAYS miniprep from FRESH culture.

Edited by Functional Screens, 27 May 2009 - 06:26 PM.


#6 eldon

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Posted 28 May 2009 - 09:33 AM

My two cents, ALWAYS miniprep from FRESH culture.


true dat.

but sometimes your schedules change on a second's notice...and there are times where a pause in a protocol still allows for sufficient results with respect to grunt work like plasmid preps.




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