Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

midi-prep


  • Please log in to reply
12 replies to this topic

#1 samita

samita

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 137 posts
5
Neutral

Posted 24 October 2013 - 08:46 AM

Hi

I did the midiprep. I precipitated the DNA with isoproponol and I can see some precipitate at the top, somehow when I try to tranfer it to the other tube for the centifugation, it disappear and after centrifugation, i did not see any pallet???? and I contniue, I try to dissolve everything in the ethanol, as I hope there is somethnig, may be i cant see it and I centifuge it again and dissolve everything in the water, I run the gel and i dont see any DNA there, Where went the DNA???

 

regards

 



#2 Kasturi Borah

Kasturi Borah

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 24 October 2013 - 08:54 AM

Add a few microlitres of NaCl and see if there is any precipitation. 



#3 samita

samita

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 137 posts
5
Neutral

Posted 24 October 2013 - 08:56 AM

how much concentration of NaCl. and add the NaCl in dry tube, suppose to have DNA pellet???



#4 Kasturi Borah

Kasturi Borah

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 24 October 2013 - 09:07 AM

After running on gel if you have saved any amounts in the tube, to it add NaCl (0.2M)



#5 samita

samita

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 137 posts
5
Neutral

Posted 24 October 2013 - 09:57 AM

I run the gel, I can see some very little amount of DNA...I measured the concentration and its 20 ng/ul. Its even low then miniprep....I dont know whats wrong happened with the  plasmid....



#6 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts
29
Excellent

Posted 24 October 2013 - 12:11 PM

Is this a kit based extraction or a crude phenol-chloroform extraction? 

 

When DNA concentrations are low, you can precipitate your DNA out with 10% NaOAc/EtOH for 30min to O/N at -80C. You also add glycogen if your ethanol solution if your DNA is being difficult.

 

Edit: Oh the typos...



#7 OA17

OA17

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 83 posts
3
Neutral

Posted 24 October 2013 - 02:46 PM

Were you able to do the midiprep of this plasmid successfully in the past or is it the first time that you are working with this plasmid?

 

Did you have any problem to grow the culture? Which antibiotic are you using? Is it ampicillin? If it's ampicilin, try changing to carbenicilin.

 

I once had problems with a plasmid at the time of doing maxipreps: the culture growed very well, but after the preparation there was no DNA or a very very  little and useless amount.

My plasmid contained a gene for ampicilin resistance, so I solved the problem just by using carbenicilin instead of ampicilin. On some occasions, when plasmids are "toxic" for the baceria, bacteria express ß-lactamase that degrades ampicilin in the culture and this way they can get rid of the plasmid, because they don't need it any more, and start growing without it. This way, when you do your midi/maxiprep you get hardly no DNA, if any. Well, more or less I think that's the way it works. Carbenicilin is less easily degraded an therefore bacteria should conserve the plasmid in order to grow.

 

Just an idea.

Good luck!



#8 samita

samita

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 137 posts
5
Neutral

Posted 25 October 2013 - 03:11 AM

the kis is based on Isoproponal precipitation, once I added the Isoproponal I can see the precipitates but they just disappear in few minutes and if I centrifuge it I dont see any pellet. Its first time I am doing the midiprep for this plasmid. Its just pET16 vector with out any gene insert into it. I just wanted to have a plasmid for my later stuff. Culture grows very well.



#9 OA17

OA17

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 83 posts
3
Neutral

Posted 25 October 2013 - 03:21 AM

Pellet disappearing might be only salts that disolve... not DNA



#10 samita

samita

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 137 posts
5
Neutral

Posted 25 October 2013 - 03:52 AM

So where is DNA then???



#11 OA17

OA17

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 83 posts
3
Neutral

Posted 25 October 2013 - 04:43 AM

In my case, this kind of problem was due to the loss of the "toxic" plasmid during culture growth (in which I used ampicilin) and that's why I didn't get any plasmid DNA after midiprep. This is the reason why I asked you which antibiotic you were using.

I don't know if this is your case, but perhaps something like this could be happening to you: bacteria grow very well in culture... but getting rid of the plasmid. Then, when you do your midiprep, you get a very low plasmid amount... in any.

 

Other possibility, of course, is that you did something wrong during the midiprep protocol, but I assume that you did everything right...

 

Anyway, good luck! :)



#12 samita

samita

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 137 posts
5
Neutral

Posted 25 October 2013 - 07:09 AM

how can I know either my plasmid is low copy number or high copy number???



#13 OA17

OA17

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 83 posts
3
Neutral

Posted 25 October 2013 - 07:42 AM

I think pET16 is a high copy number vector. You can find this information in the brochure (if it's a commercial plasmid) and/or in the internet (just google it).






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.