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*NO* DNA eluted from plasmid prep - Why?

plasmid dh5alpha lysis Zymo student

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#1 kendallncsu

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Posted 11 July 2015 - 07:44 PM

I am a research student trying to harvest DNA from a 2400bp plasmid with a ~750bp insert.  The plasmid backbone is noted as high copy.  The sample is from AddGene.

 

I prepared LB with Kanamaycin in it - it is fresh and I autoclaved before adding the antibiotic to ensure sterility.

 

I grew DH5Alpha E. coli with plasmid in LB, streaked that on a plate (also with Kanamycin), and then grew cultures from a single colony, and finally made a large overnight culture.

 

There is at least 50/50 cell volume to air, so I do not think aeration is an issue.  Also, liquid cultures are fresh - never grown for more than 24 hours.

 

I have tried completing plasmid prep kits from Zymo (I have tried Zyppy mini and ZymoPure midi), to absolutely no avail.

 

I follow the instructions meticulously, including temperature. I have tried heating buffers to increase DNA yield.  I pellet cells, resuspend in water, lyse (but not too long!), neutralize, pellet, wash the supernatant, and elute with elution buffer provided in the kit (I have not tried to elute with water).

 

UV spec DNA concentration ratios are terrible.  Just to make sure, I have also checked the eluted DNA sample in both 2% and 1.5% agarose gel stained with EtBr.  Still...nothing.  Only the ladder shows up sad.png

 

Help, please!  What is going wrong? I know that there is DNA in the bacteria...but where is it going?



#2 kendallncsu

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Posted 11 July 2015 - 07:55 PM

sorry, the backbone is 4300bp.



#3 phage434

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Posted 11 July 2015 - 08:13 PM

Make sure you let the medium cool before adding the kanamycin -- you could be destroying it. To check, inoculate a small amount of a no-plasmid E. coli strain in some of the medium and make sure it does not grow.

 

You can track the plasmid as part of your prep by saving some of the samples along the way. Following cell lysis, take a sample of the lyse cells.  Following neutralization, take another sample. Following binding to the column, take a sample of the flow through. Following washing, take a sample of the flow through. Run all of these samples on a gel, along with your final elution. The Qiagen miniprep manuals have detailed iinstructions on how to do this, but you may not need them.

 

It's possible your E. coli strain is already Kan resistant. You should test this as well.

 

A 50% full flask is not really good. Anything more than about 250 ml in a 1 liter baffled flask is poor practice. Aim for 200 ml.



#4 kendallncsu

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Posted 11 July 2015 - 09:55 PM

Hi, thanks for the suggestions.

I made sure to let the LB cool before adding the kanamycin, so I don't think the issue lies therein.

I will consult with the lab director about growing the bacteria in a larger flask as well as tracking the plasmid.

#5 kendallncsu

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Posted 11 July 2015 - 09:57 PM

And the plasmid has a kanamycin resistance gene, to clarify

#6 phage434

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Posted 12 July 2015 - 05:08 AM

I still think you should try growing your no-plasmid strain in kanamycin medium. This will check both that your medium is kiliing the strain, and that the strain is not natively kanamycin resistant.



#7 kendallncsu

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Posted 12 July 2015 - 01:34 PM

Why? Could you explain a little more please? Are you saying that I should grow Dh5alpha - with no plasmid - in kanamycin? Is this to ensure that plasmid is indeed present, rather than just antibiotic resistance from the E. Coli?

#8 phage434

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Posted 12 July 2015 - 01:48 PM

I don't know where your competent cells came from. Perhaps you are confident that they are what you think they are, and have no kanamycin resistance. But if you try to grow some untransformed cells on your kanamycin medium, and it grows, then one of two things will be true: (1) the strain is already resistant before transforming with the plasmid or (2) the kanamycin containing medium is not selecting against un-transformed cells. In either case, you have a problem. This is relevant since if you grow cells without selection, the plasmid is often lost, since non-plasmid containing cells typically grow faster and will take over a culture.

I should add that when you culture large volumes, you show grow an overnight culture of 5-10 ml and then use that culture to inoculate a large volume, and grow the large volume up in 4-6 hours. Do not overgrow it.



#9 kendallncsu

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Posted 12 July 2015 - 01:59 PM

Thank you for the clarification. I think you are certainly on to something.

Growing conditions can certainly be improved, but I think that at their current state that they should be leading to low rather than no DNA yield...provided plasmid is actually present!

I do no think that I am making procedural errors with the plasmid prep kits - I will check the kanamycin to make sure that it is truly selecting for the resistant cells and not allowing other cells to take over the culture.

One more question - is it a problem if the strain is already kanamycin resistant, or does it just make it more difficult to ascertain if plasmid is present?

Thanks for all of your ideas.

#10 phage434

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Posted 12 July 2015 - 02:10 PM

It is fatal if your non-transformed strain is kanamycin resistant. You would have no way of selecting the colonies which had been transformed from those that were un-transformed. In fact, the colonies that were not transformed would likely grow better and produce larger colonies.



#11 kendallncsu

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Posted 12 July 2015 - 02:13 PM

That makes sense. Thanks again.

#12 HawkeyeGrad

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Posted 15 July 2015 - 10:23 AM

I would suggest you always make sure that you are indeed using Kanamycin in your broth when making the plates and not ampicillin for example. You wouldn't believe just how often a simple mistake like that can happen when cloning. I would also try to do a colony PCR before growing your cells in the flask as you can test if the plasmid is there before doing clean-up etc.



#13 kendallncsu

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Posted 16 July 2015 - 06:33 PM

Hi HawkeyeGrad,

 

I set up a control situation to test the effectiveness of the Kanamycin today.

 

I put my bacteria with plasmid in LB/Kan (made fresh), put ampicillin-resistant bacteria in the same media, and then plain media in the incubator to ensure what is supposed to grow is indeed growing, and that the Kanamycin is effective in preventing unwanted growth.

 

After some research, apparently Kanamycin has a short life in solution (read: five days)...so it is probable that this is causing the issue.

 

We shall see tomorrow what the Kanamycin is and is not doing!

 

Thanks again for all of your input.







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