Absence of Supercoiling on uncut plasmid

[Cathal Garvey]

Hello all,

I'm attempting to get a stable strain of E.coli carrying a plasmid I'm working with. I've performed transformations using the Peg/MgSO4 method, selected on Ampicillin, and I have miniprepped ten candidate colonies for the plasmid.

The attached picture is an enhanced capture of the ensuing gel for candidates 2-10. Because I haven't yet ordered any restriction enzymes, I ran the minipreps as-is, expecting to see the typical three-banded pattern of relaxed/partially relaxed/supercoiled plasmid. Instead, I'm seeing only one band in each lane, which in most of the candidates corresponds to the expected 6160 base pairs.

Conditions:

• The strain is derived from a chemically competent Top10 kit, and is presumed DH10B.
• Cells were grown in LB prepared in-house (using bromelain-treated soy protein rather than casein hydrolysate/tryptone)
• Minipreps were performed using a spin-column kit, and extracted in the provided T.E.
• The gel is 1%, in-gel staining with "SafeView" from nbsbio.co.uk (with added safeview to the buffer to enhance staining)
• Gel was run at 100V to clear the wells followed by 85V.
• Illumination was by blue LEDs, filtered by orange perspex (it's a Pearlbiotech.com rig designed for sybr-safe)

NB: Safeview is designed for UV transillumination, but does excite at blue and emits green. Sensitivity is consequently less than ideal, but bands are quite crisp to the naked eye; the picture was taken with a camera that doesn't have granular enough control over exposure or focus to optimise. Even to the naked eye, there don't seem to be extra bands.

Does anyone have suggestions as to why I'm seeing apparently relaxed DNA? I am pretty confident that the DNA comes from the plasmid I used to transform: I ran negative transformations with the deionised water I've been using, and got no colonies at all. The transformed plates had plenty.

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[Dakota Hamill]

All I can offer is a google search forum post result, which may be of no use at all.

"There are three major bands produced by separating uncut plasmid DNA. In order, from slowest running to fastest running, they are relaxed or nicked circular (supercoling not yet accomplished [relaxed] or removed [nicked, a single stranded break]), linear (double stranded break), and supercoiled. There are also sometimes other forms visible: dimeric forms of the three species listed above, and sometimes, if you leave your preparation in the alkaline lysis buffer too long, there are denatured forms visible as well.

The relaxed form can be minimized by not harvesting your cells too early, the nicked form can be reduced by handling your preparations gently (this will reduce the linear form as well) and completing the plasmid recovery prep without delay. Some forms can be reduced by choosing another host (using endA- strains like DH5alpha versus endA+ strains like HB101 or BL21, for example), and formation of plasmid dimers, while not usually a big problem, is strain and vector dependent.

Maximizing the supercoiled form is always helped by working quickly.

-HomeBrew-"

From

http://www.protocol-online.org/biology-forums/posts/11517.html

Maybe the super-coiled band is just really faint?  Or like that poster mentioned maybe it's strain/vector dependent.

Cool project!  Keep us updated

[Cathal Garvey]

Hm, thanks! That's really informative, actually. It is possible that the way I'm growing and harvesting cells favours a relaxed form.

Why, I wonder, is relaxed seemingly "larger" than linear? I'd have thought it was the reverse, that relaxed-but-circular was more mobile in a gel.

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[Cathal Garvey]

Good job I've read up on non-column-based methods..

---------- Forwarded message ----------
From: Brian P Higgins <higgi...@gmail.com>
Date: 28 July 2011 18:00
Subject: Re: Absence of Supercoiling on uncut plasmid
To: Cathal Garvey <cathal...@gmail.com>


My guess is that your column cut up your plasmid.  I rarely use
columns for this very reason.  There are a ton of non-column-based
alternatives out there for plasmid isolation.  I'd try one of those
(cheap and easy).

On Thu, Jul 28, 2011 at 11:58 AM, Cathal Garvey <cathal...@gmail.com> wrote:
> Hello all,
> I'm attempting to get a stable strain of E.coli carrying a plasmid I'm
> working with. I've performed transformations using the Peg/MgSO4 method,
> selected on Ampicillin, and I have miniprepped ten candidate colonies for
> the plasmid.
>
> The attached picture is an enhanced capture of the ensuing gel for
> candidates 2-10. Because I haven't yet ordered any restriction enzymes, I
> ran the minipreps as-is, expecting to see the typical three-banded pattern
> of relaxed/partially relaxed/supercoiled plasmid. Instead, I'm seeing only
> one band in each lane, which in most of the candidates corresponds to the
> expected 6160 base pairs.
>
> Conditions:
>

>   - The strain is derived from a chemically competent Top10 kit, and is
>   presumed DH10B.
>   - Cells were grown in LB prepared in-house (using bromelain-treated soy

>   protein rather than casein hydrolysate/tryptone)

>   - Minipreps were performed using a spin-column kit, and extracted in the
>   provided T.E.
>   - The gel is 1%, in-gel staining with "SafeView" from nbsbio.co.uk (with

>   added safeview to the buffer to enhance staining)

>   - Gel was run at 100V to clear the wells followed by 85V.
>   - Illumination was by blue LEDs, filtered by orange perspex (it's a

>   Pearlbiotech.com rig designed for sybr-safe)
>
>
> NB: Safeview is designed for UV transillumination, but does excite at blue
> and emits green. Sensitivity is consequently less than ideal, but bands are
> quite crisp to the naked eye; the picture was taken with a camera that
> doesn't have granular enough control over exposure or focus to optimise.
> Even to the naked eye, there don't seem to be extra bands.
>
> Does anyone have suggestions as to why I'm seeing apparently relaxed DNA? I
> am pretty confident that the DNA comes from the plasmid I used to transform:
> I ran negative transformations with the deionised water I've been using, and
> got no colonies at all. The transformed plates had plenty.
>
> --
> letters.cunningprojects.com
> twitter.com/onetruecathal
> http://www.indiebiotech.com
>

> _______________________________________________
> Methods mailing list
> Met...@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>

[Jordan Miller]

also don't vortex or vigorously pipette or sonicate your DNA, this can introduce nicks or cuts.

also, invert tubes to mix during lysis, don't vortex or pipette or sonicate.

to mix DNA preps I usually just flick the tube with a finger a couple times and then centrifuge for ~10 sec to get everything back to the bottom of the tube.

any logistical reason why you want max supercoiled form? all you really need is a single transformed colony, right?

jordan

[kingjacob]

Cathal, can you forward any info you have on non-column-based alternatives. 

I've only ever used qiagens kits for plasmid isolation.

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Cheers,
Jacob Shiach

[Cathal Garvey]

It's not so much that I specifically want any one form over the other, at this point. I was simply suspicious; I'd been expecting to see multiple bands, and instead I see only one of the correct size for most of the plasmids. That seemed odd, given that I hadn't deliberately cut them in any way.

Could be a combination of strain + culture conditions + harvest time + extraction method. As long as the plasmid works, I don't mind much..

Jacob: Will dig up the papers and forward them on. There are two main ways of doing a miniprep these days, alkaline lysis (same as the columns but without using a column to bind the DNA) and a boiling miniprep, which is similar but uses boiling to lyse/denature everything instead of sudden pH change + detergent. I havne't yet determined whether the protocol that I'm using works, because I've only just acquired "verified" plasmid containing strains. Will try my miniprep again and if it works I'll share the buffers; should be *mostly* possible to DIY using stuff from a brewing site and the kitchen cupboard, but you'll need a centrifuge.

[Nathan McCorkle]

That gel is really out of focus! You really should have run it out
further to get more resolution in my opinion, or cut it with an
Restriction Enzyme, or PCR for confirmation.

Those bigger bands could be dimers, they look like they're slightly
above the 10kb mark... I could see them being 12kb bands.

On Thu, Jul 28, 2011 at 11:58 AM, Cathal Garvey <cathal...@gmail.com> wrote:

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[Nathan McCorkle]

Oops, forgot to comment on the coiling vs linear... super-coiled moves
the fastest, then linear, then nicked-circular AKA open-circular.
Here's a nice image and write-up:
http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/15%20electrophoresis.pdf

[Cathal Garvey]

It's a lot clearer to the eye. I think I will need to get a camera that gives me better control over focus and exposure.
The lower bands align well with the 6kb marker, but I think you're right about the upper bands eg lane 7, which aligns closer to 12kb.

On 28 Jul 2011 21:08, "Nathan McCorkle" <nmz...@gmail.com> wrote:

[Nathan McCorkle]

also only seeing one band is not worrisome to me, as the extraction
method might just be rough on the DNA, etc... as long as its
consistent and not too wasteful. ladder is usually linear, do you know
what confirmation your ladder is?

I wonder how some of these "PCR" kits select for DNA of "Plasmid"
size. I wonder how to tune that selectivity.

[Cathal Garvey]

Well, alkaline lysis is the usual way to isolate plasmids. The column is only relevant to purifying the DNA. You can equally do alcohol extraction of the clarified lysate.

It works *because* of supercoiling: supercoiled DNA is more resistant to alkaline denaturation, so it doesn't precipitate during lysis and neutralisation.

On 29 Jul 2011 09:41, "Nathan McCorkle" <nmz...@gmail.com> wrote: