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Cloning Problem (cells grow but no plasmid isolated)


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#1 Stuck with Ligation

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Posted 17 March 2010 - 12:42 PM

I am doing a difficult cloning and have to put in 3 fragments into petblue 2.

The first two fragments went in no problem however the yield decreased in the miniprep from 300 ng/uL to under 50 ng/uL (size of insert ~1200 bp).

However, the 3rd fragment is giving me massive problems. When I transform the ligated plasmid (with the 3rd insert) I get colonies like normal and the PCR screen indicated I have ~80% with the 3rd insert. When I try to grow up/streak these colonies they either refuse to grow or if they grow (in culture) no plasmid is isolated. I have done a midi prep for this plasmid multiple times and always get under 2 ng/uL (no plasmid isolated). I have run countless mini and midi preps including positive controls and they all work perfectly. What can be going on here? I have made new antibiotics thinking that it was growth of wild e. coli lacking a plasmid. Yet, when I inoculate with non-transformed cells they do not grow indicating that the antibiotic works. Also, I have done controls on the plates and they kill the non-transformed cells.


Any advice would be greatly appreciated. I am wondering if the cells can remove the plasmid. For reference the plasmid is expected to be ~5.5kb in its final size and I am using NEB 10-beta cells which are used for large plasmids and BAC's.

#2 vladooo

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Posted 23 March 2010 - 09:01 AM

Plasmid instability.
Cells can remove or alter plasmids which they don't like.
Try another kind of competent cells which might accept your plasmid.
Check the "Toxic Genes and Plasmid Instability" section of pET System Manual.

#3 Denny

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Posted 23 March 2010 - 02:34 PM

To add to vladoo's plasmid instability, are you adding glucose to 1% to all of your media? Some plasmids won't even last the night in a certain cell lines on an LB plate without glucose!

Plasmid instability.
Cells can remove or alter plasmids which they don't like.
Try another kind of competent cells which might accept your plasmid.
Check the "Toxic Genes and Plasmid Instability" section of pET System Manual.



#4 Stuck with Ligation

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Posted 24 March 2010 - 07:17 AM

No I'm not...I've tried using SURE cells and I get no colonies. I wouldn't expect this product to be toxic as analogues of it have been made in other vectors.

#5 Stuck with Ligation

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Posted 24 March 2010 - 07:46 AM

I forgot to mention I have tried increasing the antibiotic concentration on the plates and media up to 3x (300 ug/mL) of amp. and had either no growth or no plasmid if there was growth.

#6 vladooo

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Posted 24 March 2010 - 08:50 AM

One way to deal with plasmid instability is to use vecor with another antibiotic resistance than amp. Ampicilin can be degraded by beta-lactamase secreted into medium from first positive E.coli you put in liquid culture, even if it takes some time. After the amp is degraded cells can kick that hateful plasmid off and start growing without any plasmid.

#7 Stuck with Ligation

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Posted 24 March 2010 - 11:20 AM

I really don't want to switch into a new vector (I'd have to buy one and possibly re-engineer my PCR and primers). I was wondering if anyone had experience growing the cells for 3-4 hours in Amp and then adding more amp every 2-4 hours to keep the concentration high selecting for the bugs that still had the plasmid. In theory it seems like this might work.

#8 vladooo

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Posted 24 March 2010 - 01:55 PM

I understand you don't want to re-clone it into new vector. Try that glucose supplementation, use carbenicilin instead of ampicilin and subculture frequently.

#9 Denny

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Posted 24 March 2010 - 04:25 PM

Okay,
Let's review: Without knowing all the details of your protocol, I'll add a few basic tips here to cover all bases.

You've tried two cell lines, Neb Nova-10 and SURE both should work fine and as you said, analogues have not been toxic to the cells.
You've detected the presence of your gene inserted into the plasmid by PCR and then grow those positive colonies in liquid media
The cells are viable, you're getting colonies on AMP plates, those cells must contain your plasmid.
Cells are growing in liquid media with AMP, those cells must contain your plasmid.

During the culture phase a couple things may be going on here.
On the plate with amp, you can get initial growth, the plasmid can become unstable in the absence of glucose and the cells get rid of it or most of the cells get rid of it within a colony. So when you innoculate liquid culture, growth either does not happen or you get a lower amount of cells to harvest if you are not harvesting by checking the OD and just at some specified time in a protocol.

Be sure not to pick satellite colones ringing around the main colony, these satellites do not contain your plasmid. They are cells that grew after the amp got chewed up in the area around your colony. This is an indication that your plates incubated too long. When I pick colonies for PCR, I make a reporter plate when I set up my PCR and never go back to the original plate to pick colonies to grow up in liquid media. You take a gamble by going back to the original plate.

You could try a couple of simple things:

Add glucose (sterile solution) to 1% in the agar plates. Transform cells as usual.
Screen colonies by PCR and make reporter plate.

AND

Innoculate liquid media also containing glucose to 1% with positive clones.
Try this with mini preps first before ramping up to bigger quantities.
200mg/L AMP is more than plenty. Make fresh, filter sterilized stock, amp can go bad in the fridge after a while. I keep 1ml aliquots in the -20C so that it's always fresh. Only thawing what I'll use in a week or two. Shake overnight at 37C. Your cultures should be very cloudy.

OR

Innoculate the same glucose containing liquid media with 200mg/L AMP, grow for however long, till you see some growth, spin down, discard supernatant, add fresh media and fresh amp and resuspend the cell pellet. Do not just add more AMP.
The cells release b-Lactamase which hydrolyzes the amp. So you want to get rid of it by changing the media and adding new antibiotic. The article below also discusses vladoo's suggestion of carbenicilin instead of amp.
Growth will be slower so plan for that.

I'm curious about your cell density at the time of harvest as you indicate that your plasmid purification kits are working fine. If your cell density is normal, there might be a problem with the purification or may need to do a few extra things to increase recovery. (see the kit troubleshooting quide)

If you have a highly unstable plasmid, it's best to stick to the protocols, add the glucose, harvest and purify quickly. Cells in liquid media will also spit out your plasmid if they sit in the fridge too long.
Good luck, they can be a real pain sometimes at what should be the easy steps of the cloning process.

http://books.google....u...ing&f=false



No I'm not...I've tried using SURE cells and I get no colonies. I wouldn't expect this product to be toxic as analogues of it have been made in other vectors.


Edited by Denny, 24 March 2010 - 04:39 PM.


#10 Denny

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Posted 25 March 2010 - 01:23 AM

just a dumb question, but have you checked your liquid media for contamination by just incubating some media with amp and no bacteria? If not, add that to your process. :rolleyes:

#11 Stuck with Ligation

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Posted 25 March 2010 - 06:37 AM

I've checked the media and grew a tube for 3 days and still had no growth. I know the miniprep kit is fine because I ran a positive control (one of my other plasmids on the same backbone) and got similar yields to previous preps. The plates and the Amp are both brand new (under a week old).

I should check the OD's and try adding glucose but the cells I'm using lack the DE3 so they shouldn't be having any leaky expression but I realize anytime there is a start codon you can get a small amount of expression but I will setup the glucose trial next week.

#12 Denny

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Posted 25 March 2010 - 02:27 PM

Many of the cloning cell lines do not have DE3, you get that in your expression host from the bacterial DNA.

http://www.csun.edu/...c4a/petsys.html

But I have had problems in the past with cloning strains losing plasmids during culture on LB plates or liquid media in the absence of glucose. Give it a try, if it works it's a quick and easy fix.
Here's a great paper with some excellent references on plasmid stability experiments.

Low temperature and glucose enhanced T7 RNA polymerase-based plasmid stability for increasing expression of glucagon-like peptide-2 in Escherichia coli
Protein Expression and Purification
Volume 29, Issue 1, May 2003, Pages 132-139

Check out Studier's paper with recipes and nice explanations

Protein production by auto-induction in high-density shaking cultures
Protein Expression and Purification, Volume 41, Issue 1, May 2005, Pages 207-234
F. William Studier

Check your PM :lol: Your controls sound good, I'm stumped but will think on it. Good luck


I've checked the media and grew a tube for 3 days and still had no growth. I know the miniprep kit is fine because I ran a positive control (one of my other plasmids on the same backbone) and got similar yields to previous preps. The plates and the Amp are both brand new (under a week old).

I should check the OD's and try adding glucose but the cells I'm using lack the DE3 so they shouldn't be having any leaky expression but I realize anytime there is a start codon you can get a small amount of expression but I will setup the glucose trial next week.


Edited by Denny, 25 March 2010 - 02:53 PM.


#13 Stuck with Ligation

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Posted 06 April 2010 - 06:17 AM

I have tried glucose stimulation now and have had no luck. The colonies that do grow have to be recombined or have the insert remove completely because I cannot find any part of my insert by PCR. This is so frustrating. I might have to start again because I have an IRES and I think the piece of DNA that is being expressed in low levels and it is toxic. I may have to redesign my insert and use a linker.

#14 Denny

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Posted 10 April 2010 - 07:11 AM

Sounds very frustrating. Have you tried checking for your insert with restriction digest? I have had enough PCR screening fail only to find the insert is present by restriction digest, to not always trust the PCR screen. Sometimes, not often, but sometimes pcr screening doesn't work.

But, it does sound like you've got other issues and will need to do some further investigation. Bummer. When having these problems, it's a good practice to run controls every step of the way, just in case there's a problem with your methods or techniques, you can rule that out and be confident that it's not you, it's the construct and or cell line that is not cooperating!! :lol: Good luck




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