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Ligation problem: clones survive selection but nothing in there?


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#1 kingswill

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Posted 10 September 2010 - 12:32 PM

Hello!
I'm doing several ligation experiment, some are blunt-sticky and others are sticky-sticky (there are non-compatible). After ligation I transformed them into bacteria, spread them on plates, picked some, grew them in 1ml culture, mini, did restriction mapping and checked for insert.

When I run gel, I saw nothing. Blank. First I thought may be the yields were too low after mini. Confirmed by OD 260 that concentration in the mini elute were- very low, although in theory I should see them in gel (I use about 50ng-100ng for each restriction mapping).

Then I grew them up again (I kept 100 micro litre untouch from the each original 1ml culture) in 10ml culture hope to get higher yields. mini. OD260 told the yields were a bit higher than previous ones but still lower than normal mini yield. I decided carrying on the restriction mapping. Nothing in gel again. Blank.

Here is my thought:
1. mini kit may be defective. but I used this kit (I mean the columns and buffers from the same box of mini kit) many times before and they were fine. So I doubt.
2. my ampicilin in the agar plates and LB may go off. The plates were 3-4 weeks old stored at 4 degree which may be problematic. However I add fresh, whole new aliquot of Amp to the LB that I used to expand the culture. So I doubt.
3. Unless the whole lot of amp in my -20 freeze has gone off already. This could explain why I saw bacteria growing and yet got nothing from my mini. However I used this lot a month ago to grow up some plasmids and it works fine. So I doubt.
3. I kinda rule out religate vectors because 1) there are blunt-stick and non-compatible sticky-sticky ligation (I confess I didn't do dephosphrylation) and 2) If they were religate vectors I should see the vectors in gel, not completely blank.

The next thing I will do is run gel to check 1) my ligation products and 2) my mini elute. I will borrow amp from other groups, as well as mini, and do it again and see.

Do you have any suggestion about my problem?

#2 phage434

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Posted 10 September 2010 - 04:29 PM

Sounds like time for plating an untransformed control culture.
Perhaps your bacteria are resistant to ampicillin without a plasmid.

#3 HomeBrew

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Posted 10 September 2010 - 04:50 PM

Sounds like time for plating an untransformed control culture.
Perhaps your bacteria are resistant to ampicillin without a plasmid.


Exactly the thought I had. Plasmid mini-preps don't often fail, unless you screw up a step or something -- if there's plasmid in there, you'll get some; it's more good vs poor recovery, but never no recovery. Load a ton on a gel -- like 50 ul. But first, prove your recipient strain can't survive your selection conditions...

#4 kingswill

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Posted 10 September 2010 - 05:04 PM

thanks for reply!

the idea of my competent cells somehow getting resistant did come across in my mind. Yes I should spread some and see what happen.
This lot of competent cells were prepare in bulk by a previous post doc in my lab, which are very good in previous transformation and cloning and I use them for many times. They are in small aliquot and we never re-freeze and throw away once an aliquot is thawed and used. I hope it was that particular tubes that a problematic otherwise I have to throw way the whole lot and prepare again.

Just for interest, how a tube of bacteria get resistant at -80?

#5 HomeBrew

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Posted 11 September 2010 - 02:31 AM

If your recipient strain grows under selection for ampicillin resistance, there are two possibilities -- the recipient strain is in fact resistant to ampicillin, or your selection conditions are not stringent enough.

Under the "recipient strain is resistant" category, there are three possibilities: the recipient strain acquired an ampicillin resistance gene in some way, the recipient strain is not what you think it is, through a labeling error for example, or you've picked up a contaminant either during your experiment or during the preparation of the competent cells.

Under the "not stringent enough" category, there are two possibilities: the ampicillin you used has lost potency, or the media does not contain the amount of ampicillin you think it does, through improper preparation of the stock or through a dilution error, for example.

#6 kingswill

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Posted 13 September 2010 - 03:05 AM

update

Spread three plates from three tubes of competent cells. Nothing is growing. May be it's that particular tube having problem?
Wanna make sure one thing. I thaw the cells and then spread them on plates directly. Was it ok? Because when I do transformation I heat shock them and then add LB without amp and let them grow up a bit for an hour before spreading on plate. I don't know if spreading them directly will affect viability.

#7 phage434

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Posted 13 September 2010 - 04:45 AM

They should grow. But you should be thinking about how you could test this yourself. A control of spreading on a non-antibiotic plate would tell you this. This sort of dumb control becomes second nature once you start having this sort of problem and attempt to get to the bottom of it. I highly recommend the approach.

#8 HomeBrew

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Posted 13 September 2010 - 06:50 AM

If your recipient cells are sensitive to ampicillin, and your transformants are resistant yet apparently contain no plasmid after miniprep, there are two possibilities -- your plasmid is a very low-copy number plasmid and recovery via miniprep will not produce high yields, or in your transformants the plasmid has intergrated into the chromosome and is not longer replicating extra chromosomally.

If your plasmid is of the low copy number variety, and you can't see any on a gel after miniprep even after loading a large amount, then you'll have to do a maxiprep to get enough plasmid to see. What vector are you using?

If your plasmid has integrated into the chromosome, you'll have to show this by either Southern blot or by PCR using two vector-borne primers with chromosomal DNA as the template.

#9 kingswill

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Posted 13 September 2010 - 07:19 AM

thanks!

My vector is pCS2+. It's supposed to be high copy.

I think I'll do the whole experiment again starting from transformation as I have ligation products left and see what happen. I'm still not sure what is/are the causes. About the low copy number and chromosomal integration, because there are four different fragments that I'm subcloning into pCS2+ and in total tens of clones were picked, grown and mini, it's highly unlucky, if not unlikely, for all of them to suffer the same problem.

#10 HomeBrew

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Posted 13 September 2010 - 08:47 AM

I think I'll do the whole experiment again starting from transformation...


At this point, I think this is a good idea...

About the low copy number and chromosomal integration, because there are four different fragments that I'm subcloning into pCS2+ and in total tens of clones were picked, grown and mini, it's highly unlucky, if not unlikely, for all of them to suffer the same problem.


I agree this is unlikely to be the problem, but if integration is due to homologous recombination between the plasmid backbone and the recipient cell chromosome, the variety of inserts doesn't matter. Also, if you allowed an expression period of growth under non-selective conditions between transformation and plating, the number of colonies you picked is also irrelevant -- they could all be siblings of one another.

#11 kingswill

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Posted 14 September 2010 - 07:10 AM

I and my lab transformed pCS2+ constructs to the same batch of competent cells many times before and we didn't encounter this problem. I guess if the backbone contains homologous sequence to the host genome then we should see this more frequently.

it looks more and more likely that I did some mistakes during the whole process may be adding the wrong buffer during mini? Let see what happen in my second trial.

#12 phage434

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Posted 14 September 2010 - 05:54 PM

A typical failure mode is forgetting to add ethanol to the wash buffer before using it.

#13 Moppelkotze

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Posted 04 January 2012 - 04:16 AM

Dear scientists,

I hope it's ok to dig out this old topic, because I have exactly the same problem (well, I admit 3 different guys in my lab have it):

On ampicillin there are loads of colonies, but all of them (I usually analyze 30 colonies or more in this unlucky time) are empty. I analyze the clones by cracking (alkaline lysis and loaded directly on the gel - quick & dirty) and I see genomic DNA but no real plasmid (there's always a weak fluffy signal around 1,7-2 kb, which also could be RNA).

So, we decided that we have a ampicillin resistant contamination in our transformation process:

- competent E. coli: Top10 cells bought from Invitrogen (tried 3 different batches)
- DNA is not the problem, because I once transformed a plasmid I had already sequenced (so I know it was clean) and there I had colonies and received a weak signal from the right plasmid, but I assume that the contamination was also there (fluffy 2 kb signal on agarose gel after cracking)!
- LB broth autoclaved (and we also tried autoclaved + sterile filtered 0,22 um)
- heat shock 40 s 42 °C, 1 h 37 °C in LB without Amp then plated onto LB Amp agar over night 37 °C (about 16-18 h)
- final conc. of Amp: 100 µg/ml (fresh, old, -20 °C stored, plates: fresh, old, plates from other workgroups) With new Amp agar plates we have fewer colonies, or once none.

We pick the colonies from the plate with pipette tips and it's remarkably that the THING grows completely attached to the tip (no real suspension culture). Well, since I have done hundreds of cloning experiments in my past, I've seen that before, and the weiredly attached grown clone was the right one! Good ole days!

We renewed everything, we also autoclaved our pipettes! I think, we all work properly and clean, so this is definitely not the source of our trouble ../..//public/style_emoticons/default/sad.png Honestly!

Last point: with Kanamycin we do not receive any colonies any more! So the THING is slightly resistant to Amp not for Kan, but it's present in the transformation process and blocks the positive transformands out during the 1 h incubation w/o antibiotics! (that's our latest theory)

We are desperate. Please: if anyone has an idea, anything, please help us!
With best regards,
Moppelkotze and colleagues

PS: Happy new year to everybody!

#14 mdfenko

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Posted 04 January 2012 - 06:04 AM

are you sure that your ampicillin is good? prepared to the correct concentration? intact?
talent does what it can
genius does what it must
i do what i get paid to do

#15 Moppelkotze

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Posted 04 January 2012 - 06:28 AM

Hi there :)
Regarding the fact, that we don't have ANY colonies on Kanamycin, I think the problem is somewhere else. The whole transformation doesn't work anymore either on Amp or on Kan. Ampicillin is not as strong as Kanamycin, and on one hand I'm glad, we had this "old" ampicillin. So we realized it has to be a contamination!
If we would have used Kanamycin and only "good/intact" ampicillin, then ALL of our plates would have been empty empty empty for months...

But thanks for the idea. Short I can say: We tried good and bad ampicillin and on the good one, barely nothing grows. Even if we try transforming a control vector (pUC19). The plates should usually be full of positive pUC19-clones.

(final concetration is 100 µg/ml, made from a 100 mg/ml stock (-20 °C))




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