Get a plasmide - buy? share?
Mega
I was wondering where I could possibly get a plasmide like that
http://partsregistry.org/Part:BBa_K325909 ...
It can be any plasmide that has the complete lux operon, promoter and
if possible an anti-biotic ressistance.
Are there labs sending to private persons, or is there anyone to
share?
Yours sincerely
Avery louie
--Avery
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Mega
That's nearly what I've been searching for...
But it doesn't contain luciferine (?) ... :(
On 12 Dez., 07:52, Avery louie <inactiv...@gmail.com> wrote:
> As far as I know, the way you get that is by asking for the creator of the
> part to give it to you. That said carolina biological has a pglo plasmid
> with bacterial luciferase with ampicillin resistance, if thats what you are
> after.
>
> --Avery
>
Mega
Here, that's exactly what I'm looking for!!!
It would be terrific if i could get that anyway!!!
Nathan McCorkle
they've got tons of GFP or lux expression plasmids
--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Cathal Garvey
Addgene and the Parts Registry are both amazing looking resources,
crippled fundamentally by policies that punish you for not being an
academic. It bothered me to receive letters from the Parts Registry
recently asking for funding to further their goal of making SynBio more
"open" and "accessible to all" etc. etc., when I can't even request a
part as a private individual. Call me back when you update your TOS, guys.
As to your luciferase needs Mega, I can send you a wild-type strain of
P.phosphoreum which already glows in the dark, but the operon is in its
chromosome, not a plasmid. However, I might be tempted sometime soon to
hack out the operon via PCR and see if I can get it working in E.coli or
B.subtilis (not as trivial as I make it sound). There's certainly lots
of demand out there for an open-source luciferase plasmid..
Total synthesis of the operon could cost in the range of �3k: anyone
care to share their thoughts on whether that would clear kickstarter? Is
there sufficient demand to warrant a fundraiser for such a project? How
much are people willing to pay for an Open Source luciferase operon?
On 13/12/11 17:10, Nathan McCorkle wrote:
> search addgene for the gene name that you're looking for, i'm sure
> they've got tons of GFP or lux expression plasmids
--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
Nathan McCorkle
Maybe some academics that are on addgene would opensource their work... then we are done with this conversation
Sent from my mobile Android device, please excuse any typographical errors.
..but, they won't ship to non-institutional users.
Addgene and the Parts Registry are both amazing looking resources,
crippled fundamentally by policies that punish you for not being an
academic. It bothered me to receive letters from the Parts Registry
recently asking for funding to further their goal of making SynBio more
"open" and "accessible to all" etc. etc., when I can't even request a
part as a private individual. Call me back when you update your TOS, guys.
As to your luciferase needs Mega, I can send you a wild-type strain of
P.phosphoreum which already glows in the dark, but the operon is in its
chromosome, not a plasmid. However, I might be tempted sometime soon to
hack out the operon via PCR and see if I can get it working in E.coli or
B.subtilis (not as trivial as I make it sound). There's certainly lots
of demand out there for an open-source luciferase plasmid..
Total synthesis of the operon could cost in the range of €3k: anyone
care to share their thoughts on whether that would clear kickstarter? Is
there sufficient demand to warrant a fundraiser for such a project? How
much are people willing to pay for an Open Source luciferase operon?
On 13/12/11 17:10, Nathan McCorkle wrote:
> search addgene for the gene name that you're looking for, i'm sure
> they've got tons of GFP or lux expression plasmids
--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
Avery louie
Paying for things that exist and can be acquired by cleverness is somewhat silly. In any event, if y'all really want this plasmid, you can get it cheap from Carolina. And if you need more than that, I can extract it for you, provided you compensate for materials etc.
--Avery
Mega
In the next few days I try to convince a teacher at university (whom I
will be taught from in two semesters) to make the university get the
tranformation kit officially, and I pay and get it.
I think the probability is around ~30-40% to get it... But better than
nothing...
http://www.carolina.com/product/life+science/biotechnology+kits+&+materials/transformation+and+advanced+techniques/glow+in+the+dark+transformation+kit.do
(Ok, it costs 51$. But the plasmid costs ~10$, and thereby is a
manual, ampicilline (i wouldn't know where to buy it... at the
pharmacist or on the internet maybe... and some otther stuff... )
"but the operon is in its
chromosome, not a plasmid." That's surely kind of bad because you
couldn't use it further (although having glowing bacteria is very cool
for itself!!)
On 14 Dez., 02:02, Avery louie <inactiv...@gmail.com> wrote:
> Why not just buy the plasmid/get a hold of it (use your network, a lot of
> DIY bio folks are also academics), and grow up colonies and do a plasmid
> extraction? Or what about
> this<http://www.carolina.com/product/pvib+plasmid%2C+1+%26micro-g+%28200+%...>?
> Thats the plasmid you are looking for, for 10+s&h. And they will almost
> definitely ship to you/your work/somewhere official sounding. I don't
> think any one spends $50 on a plasmid and $$$ on hazmat/cold shipping when
> you have the plasmid being maintained by a stock in -80.
>
> Paying for things that exist and can be acquired by cleverness is somewhat
> silly. In any event, if y'all really want this plasmid, you can get it
> cheap from Carolina. And if you need more than that, I can extract it for
> you, provided you compensate for materials etc.
>
> --Avery
>
> On Tue, Dec 13, 2011 at 6:07 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
> > Maybe some academics that are on addgene would opensource their work...
> > then we are done with this conversation
>
> > Sent from my mobile Android device, please excuse any typographical errors.
Mega
http://www.carolina.com/product/pvib+plasmid%2C+1+%26micro-g+%28200+%26micro-l-+0.005+%26micro-g-%26micro-l%29.do?keyword=plasmid&sortby=bestMatches
plasmide is sold freely (only the transformation kit is sold to
schools etc only)
So, why don't give it a try??
I read that there is a maximum size for plasmides to use PCR (to
multiply the number of plasmides).
Will that be PCR-able??
Yours
On 14 Dez., 22:04, Mega <masterstorm...@gmail.com> wrote:
> Ok,
> In the next few days I try to convince a teacher at university (whom I
> will be taught from in two semesters) to make the university get the
> tranformation kit officially, and I pay and get it.
> I think the probability is around ~30-40% to get it... But better than
> nothing...http://www.carolina.com/product/life+science/biotechnology+kits+&+mat...
Cory Tobin
> multiply the number of plasmides).
>
> Will that be PCR-able??
The plasmid is circular DNA while PCR generates linear DNA, so you
won't be able to copy it even if it were small. The best way to copy
plasmids is just to propagate them in bacteria. When you need more
just do a miniprep.
-cory
Robert O'Callahan
competent enough. Just make sure you have an ori in there.
-Rob
Nathan McCorkle
> I just saw that the
> http://www.carolina.com/product/pvib+plasmid%2C+1+%26micro-g+%28200+%26micro-l-+0.005+%26micro-g-%26micro-l%29.do?keyword=plasmid&sortby=bestMatches
> plasmide is sold freely (only the transformation kit is sold to
> schools etc only)
>
> So, why don't give it a try??
>
> I read that there is a maximum size for plasmides to use PCR (to
> multiply the number of plasmides).
>
> Will that be PCR-able??
>
It seems like you're really jumping around with your ideas, while this
isn't bad, I think the complete kit is what you need. You need to
understand how to transform bacteria, this really doesn't have
anything to do with PCR... so PCR would be a different
activity/experiment to move on to once you first learn how to
transform bacteria.
--
Avery louie
--Avery
Mega
I just thought it would be a great idea to amplify the plasmides
before using them (to be able to use it again)
Ok, so it would make no sense to order two.... (one to make PCR and
one for transformation) because PCR cannot clone plasmides...
(understood correctly?)
When I amplify them in bacteria:
Is there a strain of e.coli with no plasmides or do I have to make gel-
electrophoresis to seperate lux-plasmides from other plasmides?
On 15 Dez., 00:40, Avery louie <inactiv...@gmail.com> wrote:
> Alternatively, read a few transformation protocols. I have had consistent
> success with CaCl transformations, and there is a post on my blog on how to
> do them. I will also be adding a protocols/media section, during my
> upcoming break.
>
> --Avery
>
> On Wed, Dec 14, 2011 at 4:58 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
> > On Wed, Dec 14, 2011 at 4:44 PM, Mega <masterstorm...@gmail.com> wrote:
> > > I just saw that the
>
> >http://www.carolina.com/product/pvib+plasmid%2C+1+%26micro-g+%28200+%...
Cathal Garvey
a dried-out strain of E.coli that you can use? Otherwise I'm sure
someone else on the list can provide you with a strain of E.coli! Check
whether there's one in the kit before asking though.
PCR can amplify plasmids, but only if you use expensive enzymes. The
cheaper enzymes aren't accurate or "processive" enough to copy long
strands of DNA. However, sometime soon I'm sure we'll have some strains
in the wild that'll make high-quality enzymes, and it'll be cheaper to
consider PCR for plasmid mass-production.
Compared to the ease of amplifying plasmids in cells though, it's almost
not worth bothering. If you've got a strain with a plasmid, and you grow
an overnight culture of them, you'll be able to extract loads of plasmid
DNA from them with a straightforward alkaline lysis plasmid prep. You
can do a prep like this without a dedicated kit if you buy all the
chemicals, and the yield of DNA is higher (I am told) if you forgo the
convenient kits and do it DIY.
As an even easier alternative to alkaline lysis, there's a way to do a
miniprep simply by boiling the cells in a special lysis buffer; it's
dirtier but quicker and easier for beginners. I'll dig out the protocol
for that if you like.
On 15/12/11 16:50, Mega wrote:
> @ Nathan,
> I just thought it would be a great idea to amplify the plasmides
> before using them (to be able to use it again)
>
> Ok, so it would make no sense to order two.... (one to make PCR and
> one for transformation) because PCR cannot clone plasmides...
> (understood correctly?)
>
>
> When I amplify them in bacteria:
> Is there a strain of e.coli with no plasmides or do I have to make gel-
> electrophoresis to seperate lux-plasmides from other plasmides?
>
>
> On 15 Dez., 00:40, Avery louie <inactiv...@gmail.com> wrote:
>> Alternatively, read a few transformation protocols. I have had consistent
>> success with CaCl transformations, and there is a post on my blog on how to
>> do them. I will also be adding a protocols/media section, during my
>> upcoming break.
>>
>> --Avery
Avery louie
How would you all feel about a DIY plasmid service where you could get ~miniprep of simple plasmids like GFP or Lux for ~$30 including s&h? I have some GFP and plenty e. coli. I would just need to grow, extract, and mail it. It would probably be at a higher concentration than the plasmid from carolina.
--Avery
Nathan McCorkle
around something ready to go.
--
Robert O'Callahan
-Rob
Nathan McCorkle
> @ Nathan,
> I just thought it would be a great idea to amplify the plasmides
plasmid is the accepted spelling, not plasmide. In chemistry the -ide
suffix has certain meaning:
http://en.wiktionary.org/wiki/-ide
> before using them (to be able to use it again)
>
Nah if you get one glowing E.coli transformed, it will grow into
billions or more in less than a day, then you can isolate the plasmid
that grew inside each E.coli cell and have billions or more plasmids,
to give to all your friends or whatever.
> Ok, so it would make no sense to order two.... (one to make PCR and
> one for transformation) because PCR cannot clone plasmides...
> (understood correctly?)
>
Just buy the kit so you have everything you need to do the
transformation, I think it will make the whole process more clear to
you.
PCR alone cannot make new plasmid, but there are techniques to combine
a PCR enzyme mix with a DNA ligase, and that would probably produce
circular DNA.... but this is far off, too far to think about now.
Learn the basics, then add complexity, you'll waste less money on
supplies that way.
>
> When I amplify them in bacteria:
> Is there a strain of e.coli with no plasmides or do I have to make gel-
You are asking if you need a strain that is 'cured' of any plasmids,
for your purposes yes you would begin with E.coli containing no
plasmids.
In some cases you may want to transform a host with multiple plasmids,
each containing different genes and different selection systems
(usually antibiotics are used for selective pressure)
> electrophoresis to seperate lux-plasmides from other plasmides?
>
To 'cure' a host of a plasmid, you just grow it for many generations
with selective pressure needed to maintain the plasmid. So if the
plasmid had gfp and amp genes, you would select with ampicillin in the
growth media, to cure the strain you just keep growing but don't add
the ampicillin.
Mega
dirtier but quicker and easier for beginners"
That sounds great
"plasmide-plasmid"
well, didn't know that... I'm german speaking, and one 'rule' is when
you have a german-latine derived word just add a -e and you have the
english one. It's true for 90% of the cases...
I asked my proffesor at university if he knew where to get e.coli lab
strains.... He recommended me another professor, and added that we may
even have a strain at university .
Do lab strains have no plasmids in general? I don't think so...
___
Well I looked at the transformation kit and its 'ingredients'...
* E. coli
* Plasmid DNA Solution
* Transformation Buffer and Luria Broth
* Ready-to-Pour Plates (Demonstration Kit contains prepoured
plates)
* Ampicillin (not in Demonstration Kit)
* Spreading Beads and Sterile Tubes
* Pipets and Transfer Loops
* Teacher's Manual with Student Master Sheets
I may get CaCl at the drug store/pharmacist or produce it myself
(CaCO3+HCl; Ca(OH)2 + HCl)
E. Coli is alo available anyhow (although it may have plasmids, which
would not be good)
Ampicillin - maybe at the drug store.
Agar yes, (LB Agar? - could i substitut that by agar+sugar+ usual
instant soup)
And i know someone who has conducted heat shock at his university. He
lives 100 km away from me, but he surely would help me when I have a
plasmid and the other ingredients.
Cathal Garvey
beats the hell out of the CaCl method for most applications.
Also, your university almost certainly has lab strains without plasmids,
and almost all basic lab strains are plasmid free (although most people
promptly add plasmids to get their experiments done!).
You can get ampicillin and other selective antibiotics from
NBSbio.co.uk, although be careful to order by *email*, not online, and
specifically tell them to charge you VAT! Otherwise, it causes a mess,
because they assume their customers are VAT registered and bill you
without VAT first, then realise you're not VAT registered, and need to
invoice you again...
Still, nbsbio.co.uk are a great, friendly company with a great selection
of things you'll need in small enough quantities to be affordable. Just
check before buying that they don't stock another brand that's cheaper;
they offer several brands of many products, and the difference in price
can be very large.
Also, for microbiology equipment check out www.brouwland.com. Don't omit
the "www.", because their website is faulty.
Mega
plasmids,
and almost all basic lab strains are plasmid free (although most
people
promptly add plasmids to get their experiments done!)."
You're sure of this?
Because I don't know exactly whether or not our university really
conducts genetic engineering... But in each case it's by far not the
main topic of the faculty...
But if the teacher who is in charge of the lab equipment (whom I asked
via email) says 'yes we have them' how can I know if there's really
no plasmid in? Because we're not specialised in g.e., he my not know
that... (?)
On 16 Dez., 15:27, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Allow me to recommend using the PEG/MgSO4 method once more. It really
> beats the hell out of the CaCl method for most applications.
>
> Also, your university almost certainly has lab strains without plasmids,
> and almost all basic lab strains are plasmid free (although most people
> promptly add plasmids to get their experiments done!).
>
> You can get ampicillin and other selective antibiotics from
> NBSbio.co.uk, although be careful to order by *email*, not online, and
> specifically tell them to charge you VAT! Otherwise, it causes a mess,
> because they assume their customers are VAT registered and bill you
> without VAT first, then realise you're not VAT registered, and need to
> invoice you again...
>
> Still, nbsbio.co.uk are a great, friendly company with a great selection
> of things you'll need in small enough quantities to be affordable. Just
> check before buying that they don't stock another brand that's cheaper;
> they offer several brands of many products, and the difference in price
> can be very large.
>
> Also, for microbiology equipment check outwww.brouwland.com. Don't omit
Mega
I just need PEG and MgSO4 (no CaCl)?
No centrifuge, no ice and no hot water?
When I asked @Carolina via 'contact function' if they could send it to
Europe, there was written answer will arrive within 24 hours. That
should have been yesterday in the evening.
Maybe the request got lost, they are extensively searching for a
delivery service or nobody has yet read my request...
Cathal Garvey
the DNA.
The protocol in brief (I'm writing a fuller, prettier version for
inclusion in the "Cookbook" :D ):
1) Prepare transformation medium using 2g PEG-3350 (Miralax, GoLytely,
GlycoLax, etc.), 0.55g MgSO4 (Epsom Salt), 6mls broth; bring to 10mls
with more broth when everything is fully dissolved, and sterilise. Then
keep chilled until use.
2) Using same broth, start a new culture and incubate it at 30/37
celcius for 3-5 hours until it starts to get turbid (cloudy due to cell
growth).
3) Take 500mls of cells from the active culture, mix 50/50 with chilled
transformation medium, leave on ice for 10 minutes.
4) Add some plasmid DNA and stir gently with pipette tip to mix. Leave
on ice another 30 minutes.
5) Incubate samples for 30 minutes at 30/37C to allow expression of
resistance genes to begin before plating.
6) Plate the cells on selective media; agar containing whatever
antibiotic your plasmid gives the cells resistance towards.
7) If you get colonies the next day, pick ten (10!) to test for your
plasmid; there are frequently random mutants or bacteria containing
malformed plasmids, so you want to be certain that the bugs have the
plasmid you want. Culture each colony in its own broth culture, 5mls,
and the next day miniprep 4mls of them to extract plasmid DNA. Then run
all of the samples on a gel with the original DNA beside them, and only
keep a culture that contains a plasmid of similar size.
Recommendations:
- Make two plates per sample using different amounts of the transformed
cells, in case you get too many or too few cells on one plate. 10uL and
100uL, or 100uL and 1ml may be appropriate.
- Plate a positive and negative sample if possible, to rule out errors
later. That way, if things go wrong and you ask for help, you'll have
two or three "controls" to compare against, and it will be easier to
find the problem.
- For a positive, you'll need a culture of cells that are already
resistant to the antibiotic, perhaps an older culture of pre-transformed
cells if you have any.
- For a negative, subject some cells to the exact same procedure, but
add water instead of DNA at step 4.
- You might be wise to also plate some cells from step 3 that have
*not* been exposed to the transformation medium, and plate those.
Hope that helps!
Cathal
On 17/12/11 12:03, Mega wrote:
> When using the PEG/MgSO4-Methode,
> I just need PEG and MgSO4 (no CaCl)?
> No centrifuge, no ice and no hot water?
>
>
>
> When I asked @Carolina via 'contact function' if they could send it to
> Europe, there was written answer will arrive within 24 hours. That
> should have been yesterday in the evening.
> Maybe the request got lost, they are extensively searching for a
> delivery service or nobody has yet read my request...
--
Mega
Now the only thing I need is E. coli without plasmids (lab strain) and
the plasmid.
I can't await the moment when the answer e-mail from Carolina
reguarding pVIB plasmid arrives.
John Griessen
> Hope that helps!
> Cathal
that was a really fine description of your cookbook, I mean protocol,
for growing and isolating bugs of a certain kind and I really really
liked hearing about the "plate ten, yes, ten!" so you can skip mutants
and random noise. Thanks for a peek at this by an armchair bio
thought experimenter.
After I get some gear out there and selling, I may just use some of it!
John
Mega
Now I just need E.Coli without any plasmides to replicate the pVIB.
As I wrote, one of my teacher agreed to get me a lab strain of e.coli
for private research. But how can I know (or better verify) there's no
plasmid in it??
Cathal Garvey
Minipreps can be done with spin-column kits, or you can roll-your-own.
Here are some guides that might be helpful:
Boiling Miniprep:
http://bioteachnology.com/plasmid-dna/rapid-boiling-method-plasmid-dna-extraction
Alkaline Lysis Miniprep:
http://bioteachnology.com/plasmid-dna/plasmid-dna-extraction-alkaline-lysis-method
My poorly tested (read: basically untested so far, sorry) take on
Alkaline Lysis:
============================
This method is commonly used to extract plasmid DNA from bacteria.
All Centrifugation is performed at 8Krcf-10Krcf. Use Deionised H20 for
everything.
Buffer I: 0.2g Lysozyme, 0.9g Glucose, 0.3g EDTA, 0.3g Tris to 100ml
with H20
Buffer II: 0.8g NaOH, 1ml SDS, to 100ml with H20
Buffer III: 24.6g NaAc, Adjust to pH4.8 with Acetic Acid, to 100ml with H20
Buffer NaTH: 0.82g NaAc, 0.606g Tris, to pH8 with Acetic Acid, to 100ml
with H20
Buffer TE: 1.21g Tris, 0.3g EDTA, Adjust to pH8 with Acetic Acid, to 1L
with H20
Step 1: Isolate cells from 0.5ml broth, grown overnight, by
centrifugation for 1m
Step 2: Remove liquid phase, resuspend cell pellet in 0.1ml Buffer I.
E.coli: Incubate at 0C for 30 minutes. B.subtilis: Incubate at 37C for
30 minutes.
Step 3: Add 0.2ml of Buffer 2, cap & invert x10 to mix thoroughly.
Incubate 0C for 5m.
Step 4: Add 0.15ml of Buffer 3, cap & invert x10 to mix thoroughly.
Incubate 0C for 60m.
Step 5: Centrifuge for 10m. Carefully remove 0.4ml of clear liquid phase
to new tube.
Step 6: Discard solids. To liquid, add 2.25ml 70% Isopropanol. Incubate
-20C for 20m.
Step 7: Centrifuge for 10m. Carefully remove liquid, leave clear/white
DNA pellet.
Step 8: Resuspend in 0.1ml Buffer NaTH, add 0.2ml Ethanol/Isopropanol.
Incubate -20C for 10m.
============================
Mac Cowell
Cathal Garvey
and I can't seem to find either on my HDD just now.
I'd suggest 200ul-400ul, with 20ul-40ul DNA, off the top of my head.
On 20/12/11 16:01, Mac Cowell wrote:
> What volume of cells is used in step 3?
>
> 231.313.9062 // @100ideas // sent from my rotary phone
Avery louie
Mac, I would check the current protocols book. Im pretty sure it's in there.
--Avery
Cory Tobin
> and I can't seem to find either on my HDD just now.
>
> I'd suggest 200ul-400ul, with 20ul-40ul DNA, off the top of my head.
Here's the paper. Full text is free on Pubmed Central.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC286873/?tool=pmcentrez&rendertype=abstract
Looks like they used 0.1mL of cells in TSS and 100pg of DNA. But
since there is no heating step, any volume of cells should work just
fine.
-cory
Mega
(-20°C)?
Or do I have to store them at -80°C?
Do I have to add something to the plasmids so they don't get damaged
by freezing??
Avery louie
-20 is fine, and you don't need to add anything.
--A
Avery louie
--AL
Cory Tobin
> YMMV, but it should be a fine place to store a plasmid.
Just remember that most kitchen freezers have a defrost cycle. If you
want you sample kept consistently at -20 you would need to use a
non-defrosting freezer.
-cory
Simon Quellen Field
Cory Tobin
> there would otherwise be empty air) then the freezer will have a large
> thermal mass, and the temperature of the contents will stay within a
> half degree Celsius of the temperature you have set it, even during
> defrost cycles.
Ah, good point.
Cathal Garvey
freezer, surround with thermal mass, and leave it alone.
However, if you'll be using the DNA within the month, it's probably best
*not* to freeze it..*if* it comes in "Tris-EDTA" buffer. This is
commonly shortened to TE. TE buffer protects DNA from nucleases and
maintains a pH that enhances stability for extended periods at 4C
instead; no freezing required.
The reason to opt for no-freeze for short periods is that DNA is harmed
by freeze and thaw, so you don't want to be freezing it today only to be
thawing it for use tomorrow.
Mega
(Because the plasmids are 200ul). Is that size correct, or shall I get
another one of the sizes below?
(I hope to be able to use it for all kind of experiments with
bacteria. Would be 10-100ul better?
0.1-2.5μl
0.5-10μl
2-20μl
5-50μl
10-100μl
20-200μl
50-200μl
100-1000μl
200-1000μl
1000-5000μl
Cathal Garvey
recommend 2-20uL, because you can always pipette a few times to reach
60uL or 80uL. Being careful not to touch the destination fluid before
returning to the source fluid, of course; that would be
back-contamination. Bad news for things like PCR.
On 22/12/11 10:20, Mega wrote:
> I just ordered an adjustable pipette in the range of 100-1000 ul.
> (Because the plasmids are 200ul). Is that size correct, or shall I get
> another one of the sizes below?
> (I hope to be able to use it for all kind of experiments with
> bacteria. Would be 10-100ul better?
>
> 0.1-2.5οΏ½l
> 0.5-10οΏ½l
> 2-20οΏ½l
> 5-50οΏ½l
> 10-100οΏ½l
> 20-200οΏ½l
> 50-200οΏ½l
> 100-1000οΏ½l
> 200-1000οΏ½l
> 1000-5000οΏ½l
>
>
>
>
>
>
>
> On 22 Dez., 10:19, Cathal Garvey <cathalgar...@gmail.com> wrote:
>> As with what Averie said, freezing is easy with DNA. Just put it in the
>> freezer, surround with thermal mass, and leave it alone.
>>
>> However, if you'll be using the DNA within the month, it's probably best
>> *not* to freeze it..*if* it comes in "Tris-EDTA" buffer. This is
>> commonly shortened to TE. TE buffer protects DNA from nucleases and
>> maintains a pH that enhances stability for extended periods at 4C
>> instead; no freezing required.
>>
>> The reason to opt for no-freeze for short periods is that DNA is harmed
>> by freeze and thaw, so you don't want to be freezing it today only to be
>> thawing it for use tomorrow.
>>
>> On 21/12/11 22:39, Mega wrote:
>>
>>> When I get the plasmids, how long can I store them in the freezer
>>> (-20 C)?
>>> Or do I have to store them at -80 C?
>>
>>> Do I have to add something to the plasmids so they don't get damaged
>>> by freezing??
>>
>> --www.indiebiotech.com
>> twitter.com/onetruecathal
>> joindiaspora.com/u/cathalgarvey
>> PGP Public Key:http://bit.ly/CathalGKey
>
jlund256
samples. Samples kept in a standard freezer should be placed inside
an air tight outer container--something with a screw on lid or a
plastic container that seals well.
Jim Lund
Simon Quellen Field
Ethan
first post here.
Thank you all for the valuable information in this thread. I am still
trying to get my lab set up to do basic transformations. One thing I
am curious about is purification of plasmids so that I can continue to
use the plasmids that I have. Looking for miniprep columns online, I
am unable to locate a seller that ships to residential addresses. Is
there a way to circumvent that? Also, specifically to Cathal Garvey,
you mentioned that you could "roll your own" in the context of
columns. Do you have any more information on this? I would like to
keep costs to a minimum, so making my own equipment is definitely of
interest to me.
Additionally, I have a general question about transformations (I have
done a few before, but I didn't have full details on the strains of
bacteria at the time). When performing a transformation, is it
necessary to work with a strain that is deficient in restriction
modification, or is that only an issue if you are working with a fully
synthetic sequence (i.e. just purchased from a DNA synthesis company)?
Thank you, everyone!
Cathal Garvey
Great to hear you're getting into transformations. I hope you aren't
waiting too long until your first results.
As to "Rolling your own" minipreps, you actually don't need columns at
all. Indeed, I am told anecdotally that purifying without columns gives
far greater yields!
Instead of using Columns, you can purify without by precipitating the
plasmid DNA using alcohol, centrifuging the DNA to the bottom of an
eppendorf tube after precipitation, and washing by resuspending in an
alcohol-based wash buffer followed by another resuspension.
Look at it this way; the columns simply offer a nice place for the DNA
to precipitate, and hold onto the precipitated DNA while you wash it.
They "release" DNA when you rinse them with a buffer that can
successfully dissolve it away. You don't need this nice place, if you
just use centrifugation to keep the DNA where you can handle it while
removing buffers.
The hard part without a column is drying the DNA free of alcohol, but
not *too* dry (because it's harder to redissolve), and then dissolving
it again. If it's been dried for not-long-enough, it will have residual
alcohol in it which might affect freezing, reactions, or even its
ability to dissolve. If it's too dry, you might need to leave it
overnight to redissolve correctly!
In any case, you can get columns and miniprep kits, along with a bunch
of other stuff, from NBSbio.co.uk if you live in Europe. Just make sure
to *order by email*, and tell them that you are *not* VAT registered.
They've always shipped to me without hassle, and are very friendly and
professional.
On transformations and enzymes; it massively increases transformation
efficiency if your cells have no restriction/modification systems, but
it won't entirely prevent it. My favourite culture, B.subtilis 168, has
an enzyme that cuts similarly to XhoI; if there are any XhoI sites
present, your efficiencies are 100 times lower. Once transformed though,
the methylase kicks in and the DNA is stable, and can be transformed
back in after a miniprep without problems (but not after PCR, because
PCR doesn't methylate!).
For difficult transformations, many companies offer methylases to
protect the DNA before transformations or reactions. I've never used
them, and I imagine they are a pain in the arse to use.
If you *are* in Europe, we'll have to meet sometime at a summit to talk
transformations! If not, we'll surely get a chance anyway eventually. :)
On 22/12/11 01:45, Ethan wrote:
> First off, greetings! I have been lurking for a bit, but this is my
> first post here.
>
> Thank you all for the valuable information in this thread. I am still
> trying to get my lab set up to do basic transformations. One thing I
> am curious about is purification of plasmids so that I can continue to
> use the plasmids that I have. Looking for miniprep columns online, I
> am unable to locate a seller that ships to residential addresses. Is
> there a way to circumvent that? Also, specifically to Cathal Garvey,
> you mentioned that you could "roll your own" in the context of
> columns. Do you have any more information on this? I would like to
> keep costs to a minimum, so making my own equipment is definitely of
> interest to me.
>
> Additionally, I have a general question about transformations (I have
> done a few before, but I didn't have full details on the strains of
> bacteria at the time). When performing a transformation, is it
> necessary to work with a strain that is deficient in restriction
> modification, or is that only an issue if you are working with a fully
> synthetic sequence (i.e. just purchased from a DNA synthesis company)?
> Thank you, everyone!
Mega
Mega
That seems quite easier than an alkaline lysis, because the only
chemical substance I need is STET-buffer. And this one substance I'll
get anyhow.
The supernatant 'll be junk and within the liquid there will be the
plasmids (and some proteins that won't affect further transformations
very much) then?
____
Merry Christmas @ all !!
Cathal Garvey
to collect for plasmids. The pellet, meanwhile, is all the stuff that's
formed from precipitation of cellular matter.
To isolate the DNA, you will also need alcohol and perhaps sodium
acetate. For alcohol, you can either use ethanol (if you can get pure
ethanol where you live) or isopropanol (available anywhere). You need
different amounts of whichever one you use, and the precipitated DNA
will look a little different (white with ethanol, glass-like with
Isopropanol), but the DNA will be fine either way.
If you need sodium acetate (I can't remember, check the protocol), you
can either make it by reacting acetic acid and sodium carbonate (vinegar
and baking soda), or more preferably buy it online from a pure chemicals
website such as mistralie.co.uk.
Alternatively, those re-usable hand warmers that contain a little metal
tag you bend to activate them? Which you boil to re-melt for reuse? That
liquid is sodium acetate trihydrate, so you can use it provided you
calculate the amounts including the extra water (you'll find a
comparison of the molar weights on wikipedia), and you should use it as
a *solid*, not a liquid. The liquid form may spontaneously crystallise
(I think?) on contact with other stuff like leftover cell matter,
releasing tons of heat and inconveniencing your experiment. :)
Mega
(97%). I think isopropanol is more difficult to obtain for me.
Jeswin
> In our university's lab there are big ammounts of 'pure' ethanol
> (97%). I think isopropanol is more difficult to obtain for me.
>
percentage, above 70% and maybe 80% or 90%, at your local drugstore. I
would think it it is much harder to procure ethanol.
Cathal Garvey
You may want to search for 'pure' salt though, without iodine, preservatives or anticaking agents, as these may affect the outcome.iodine in particular would strike me as a dodgy thing to add to a DNA prep, it can be pretty reactive right?
Jeswin <phill...@gmail.com> wrote:
>--
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www.indiebiotech.com
Simon Quellen Field
Cathal Garvey
Mega
STET-Buffer
lysozyme (really necessary??)
water
isopropanol
Ethan
the lysozyme. From my understanding, its purpose is to break down
parts of the bacterial cell wall (namely peptidoglycan). I imagine
that eliminating it from the procedure would cause either a drastic
drop in yield or total lack of yield.
I am looking into several reagents, and I was wondering if there is
any functional difference between the various salts of EDTA available.
My guess would be that the disodium and tetrasodium salts would only
differ in the dissolved pH, and so might need additional adjusting
accordingly. However, having a calcium salt of EDTA seems
counterintuitive to me because it functions to chelate several ions in
solution, including Ca2+. Which salt is typically used in the
laboratory? Thanks!
Cathal Garvey
E.coli, but it *is* recommended. I suggest getting oodles of cheap
lysozyme from www.brouwland.com, where you can get it as a crystallised
powder for far, far cheaper than commercial biotech grade lysozyme. It
won't be as pure I expect, but that's not really necessary as far as
stage 1 of a miniprep is concerned. With some tweaking/trial/error you
should be able to find an amount that works just as well for less than a
hundredth of the price.
I'd stick with sodium salts of EDTA, but it might be worth getting the
lower sodium contents as you can use this more easily with DIY
electrophoresis buffers, too. The more sodium in your EP buffers, the
more current and the more heat. In the original paper on using Sodium
Borate, the authors dedicated part of their rant-against-TAE/TBE to the
sodium content of the dissolved EDTA.
--
Nathan McCorkle
http://bitesizebio.com/articles/a-menagerie-of-mini-prep-methods/
Sent from my mobile Android device, please excuse any typographical errors.
Mega
Have a look at that: http://www.youtube.com/watch?v=TZeL7WIkQC0
He just uses a dish detergent and sodiumhydroxide (both of which I
already have at home) and isopropanol to conduct an alkaline -home-
miniprep!!
I immagine that the purity of the plasmids is not very high, maybe
some 80-85% ??
But anyway, is it neccessary for a transformaiton to have relatively
pure plasmids? Some proteins and other substances (e.g. luciferin,
sugar, etc.) that are in the solution don't affect the would-be
transformed bacteria, thay may consume them or not. No big deal?
But, what happens to the chromosomal DNA in this process? Is it
denatureted by the dish detergent??
Ethan
at that. The majority of the DNA isolated in that prep was probably
nucleoid and not plasmid. I am not completely sure, but I would
estimate that the plasmid content would be very low. One thing that I
would worry about in his procedure would be nuclease activity. Using
something like TE buffer instead of water should help to deactivate
them before they chew up all the DNA. The chromosomal DNA here is a
large part of what is ultimately extracted. The dish detergent mostly
serves to disrupt the lipid membranes/lyse the cells.
For a plain old transformation, the plasmids don't have to be
extremely pure, but I imagine efficiency would drop off at a certain
point. However if you plan to do any analysis (electrophoresis,
sequencing, etc) or engineering on the plasmid, then you would need
the most pure form available. In this
Mega
boiling lysis??
(I'll try to get the chemicals now... Just STET, isopropanol/ethanol
and water?)
On 27 Dez., 18:13, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Correction: 300mM. I blame Christmas fatigue for my unreliable literacy.
>
>
>
> Cathal Garvey <cathalgar...@gmail.com> wrote:
> >By the way, a reliable source tells me that 100mM of sodium chloride is
> >just as good as sodium acetate. As table salt is cheaper than
> >handwarmer filling, this is helpful to know.
>
> >You may want to search for 'pure' salt though, without iodine,
> >preservatives or anticaking agents, as these may affect the
> >outcome.iodine in particular would strike me as a dodgy thing to add to
> >a DNA prep, it can be pretty reactive right?
>
>
> >>On Tue, Dec 27, 2011 at 9:55 AM, Mega <masterstorm...@gmail.com>
Mega
Ok, so its not that easy.... It's a pitty!
I won't need the chromosomal DNA & nuclease digesting my plasmids is
also terrible...
Nathan McCorkle
An alkaline miniprep is basically an acid/base (A/B) extraction where the point is to "defat" the cell solution using detergent to act as the nonpolar component... it diverges from a normal A/B extraction because you don't separate nonpolar from polar before neutralizing... thanks to the size of chromosomes/nucleosomes they get caught up with the nonpolar stuff and pellet out.
Lysozyme isn't needed, but helps. Sugar in buffer is probably just for osmolarity so cells don't burst before being alkalinized, because the alkalinity deactivates nucleases. Triton-X is a line of fancy and well documented detergents, so protocols have been refined to make sure its all cleaned from the plasmid solution... other detergents will work, but the protocol may be lossy without lots of experimental optimization. EDTA is also there to stop nuclease activity but again not absolutely required... unless you plan on storing the plasmid for a while or at room temperature.
Sent from my mobile Android device, please excuse any typographical errors.
Cathal Garvey
isolation is the fine balance of alkalinity between "denatures DNA" and
"Denatures *even supercoiled DNA*". Because plasmids are usually found
in some form of supercoiling within cells, they are tightly wound up and
resistant to denaturing. The high pH of the lysis/denaturing buffers in
minipreps force protein, fat *and* chromosomal DNA into an insoluble
mass which can be easily removed, while leaving plasmids intact.
So, provided you could precisely hit the target pH in one swift step,
you could do the miniprep with just Sodium Hydroxide as in the youtube
video. However, that would be pretty prone to error!
Also, you can do without lysozyme for lab strains of E.coli, and other
gram negatives may burst just because of the high pH. However, I
wouldn't rely on that to work with gram positives; they have just too
much peptidoglycan to burst in the needed time frame. By the time the
sodium/potassium hydroxide chewed them open, it would probably have
damaged the DNA quite a bit also.
Lysozyme's cheap to buy from brew-shops, and you can prepare it from
eggs using cheap vodka, do might as well use lysozyme. If you're
prepping your own, only use free-range eggs; not only is it nicer to the
hens, but they tend to have far more lysozyme in them.
--
Mega
instruction about this?
Do I just have to mix egg (the yellow and the white piece - both of
them?) with ethanol and kind of 'shake' it (using a kitchen
machine)?
Aren't there any other proteins/enzymes that will be soluted by the
ethanol??
Cathal Garvey
www.brouwland.com - But if you really want to, it goes roughly like this:
1) Mix 10mls egg *white* with 30mls Vodka (assuming 40% ethanol vodka:
you are aiming for a final egg/vodka ethanol concentration of 30%)
2) Leave at room temperature for about 3 hours.
3) Dilute in water at least 50/50 to stop precipitation of further
protein from occurring.
4) Filter out all the crap using a coffee filter
5) Dry it out gently, perhaps on a wide, flat, very clean pan.
6) Collect crystallised residue, which should have lysozyme activity.
To determine how *much* lysozyme you've got, you may have to standardise
your batches by adding some in different concentrations to
just-inoculated batch cultures of a susceptible bacterium, and
standardising against the minimal inhibitory concentration. In other
words, for each batch, make five or so different dilutions of the powder
and add 1ml of each dilution to a 9ml potato-dextrose-broth culture
you've just spiked with a fresh overnight culture of B.subtilis or the like.
You could also use freshly spiked yoghurt, as both relevant cultures in
natural yoghurt are gram-positive and should be susceptible to lysozyme.
Bear in mind that while this protocol is based upon genuine literature
hunting, I've never tested the resulting lysozyme. Like the agarose
misadventure, it was a proof-of-principal thing for me: I just wanted to
know that I *could* have lysozyme if I really needed it and supply
sources dried up! :)
On 02/01/12 11:43, Mega wrote:
> Lysozyme from eggs? that sounds great!! Do you have a protocol/
> instruction about this?
>
> Do I just have to mix egg (the yellow and the white piece - both of
> them?) with ethanol and kind of 'shake' it (using a kitchen
> machine)?
> Aren't there any other proteins/enzymes that will be soluted by the
> ethanol??
--
Mega
"dsm 1103". how can i find out if it has plasmids inside? on google i
find some related articels but no gene map or description of the
strain (i'm looking for a kind of datasheet if you will)
best regards
On 2 Jan., 14:24, Cathal Garvey <cathalgar...@gmail.com> wrote:
> It's messy, so you're far better off buying your lysozyme fromwww.brouwland.com- But if you really want to, it goes roughly like this:
Cathal Garvey
would surely mention if the strain carried a plasmid already. If the
papers involve transformation of new plasmids *into* dsm 1103, then you
can be pretty sure there aren't already plasmids in it unless they are
specifically mentioned.
Really though, wouldn't the person you got the strain from be able to
tell you things like this offhand?
On 16/01/12 11:25, Mega wrote:
> dsm 1103
Cathal Garvey
E.coli "dsm 1103"
I immediately found the following sources of strain-specific information:
http://bccm.belspo.be/db/lmg_strain_details.php?NUM=8223&COLTYPE=
and
http://www.straininfo.net/strains/52432/browser;jsessionid=BB0E5C1E1B14A2ED34725352A076411F
(via: http://www.straininfo.net/strains/134267 )
Learn to use quotes in search engines, and you'll get much better
results with specific, multi-word queries like strain IDs.
Some notes I get from speed-reading the info in the first link; At least
somebody's classification system thinks this strain qualifies as
Biohazard Class/Level 2, which means you probably can't culture this
legally outside a Class 2 lab. Most lab strains of E.coli are Class 1,
meaning that most places have no laws requiring anything specific.
If this strain is Class 2 where you live, or if there's a good reason
why anyone thinks it's Class 2, perhaps you should look for another
strain. Given that a strain comes for free with the pGlo kit AFAIK, I'd
stick with that rather than this strain.
Apparently it is known to make Alpha-Hemolysin:
https://en.wikipedia.org/wiki/Haemolysin_A - Which indicates that this
strain is at least a little bit pathogenic. I recommend throwing it away
and getting a less hazardous culture; ideally you're looking for E.coli
K12 or something descended from K12. Nice cultures also include
"DH5-Alpha" or "DH10-Beta" (AKA "Top-10")
On 16/01/12 11:25, Mega wrote:
Mega
links (( http://bccm.belspo.be/db/lmg_strain_details.php?NUM=8223&COLTYPE=
and
http://www.straininfo.net/strains/52432/browser;jsessionid=BB0E5C1E1B...
))
Maybe he didn't know?
I don't think it would be very smart to work with potential
pathogenes, so I may have to dispose of them.... Maybe he feels sorry
and gets me another strain :)
On 16 Jan., 14:47, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Sod it, I went to duckduckgo.com and searched for:
> E.coli "dsm 1103"
>
> I immediately found the following sources of strain-specific information:http://bccm.belspo.be/db/lmg_strain_details.php?NUM=8223&COLTYPE=
> (via:http://www.straininfo.net/strains/134267)
> Learn to use quotes in search engines, and you'll get much better
> results with specific, multi-word queries like strain IDs.
>
> Some notes I get from speed-reading the info in the first link; At least
> somebody's classification system thinks this strain qualifies as
> Biohazard Class/Level 2, which means you probably can't culture this
> legally outside a Class 2 lab. Most lab strains of E.coli are Class 1,
> meaning that most places have no laws requiring anything specific.
>
> If this strain is Class 2 where you live, or if there's a good reason
> why anyone thinks it's Class 2, perhaps you should look for another
> strain. Given that a strain comes for free with the pGlo kit AFAIK, I'd
> stick with that rather than this strain.
>
> and getting a less hazardous culture; ideally you're looking for E.coli
> K12 or something descended from K12. Nice cultures also include
> "DH5-Alpha" or "DH10-Beta" (AKA "Top-10")
>
> On 16/01/12 11:25, Mega wrote:
>
>
>
>
>
>
>
>
>
> > I finally got non-pathogenic e.coli from a proffessor. it's the strain
> > "dsm 1103". how can i find out if it has plasmids inside? on google i
> > find some related articels but no gene map or description of the
> > strain (i'm looking for a kind of datasheet if you will)
>
> > best regards
>
> > On 2 Jan., 14:24, Cathal Garvey <cathalgar...@gmail.com> wrote:
Mega
gets somewhere [in the human body] where it doesn`t belong " so
what to do?
On 16 Jan., 17:44, Mega <masterstorm...@gmail.com> wrote:
> Well, i asked my professor via e-mail about that and sent him those
> links ((http://bccm.belspo.be/db/lmg_strain_details.php?NUM=8223&COLTYPE=
>
> Maybe he didn't know?
>
> I don't think it would be very smart to work with potential
> pathogenes, so I may have to dispose of them.... Maybe he feels sorry
> and gets me another strain :)
>
> On 16 Jan., 14:47, Cathal Garvey <cathalgar...@gmail.com> wrote:
>
>
>
> > Sod it, I went to duckduckgo.com and searched for:
> > E.coli "dsm 1103"
>
> > I immediately found the following sources of strain-specific information:http://bccm.belspo.be/db/lmg_strain_details.php?NUM=8223&COLTYPE=
> > andhttp://www.straininfo.net/strains/52432/browser;jsessionid=BB0E5C1E1B...
> > (via:http://www.straininfo.net/strains/134267)
>
> > Learn to use quotes in search engines, and you'll get much better
> > results with specific, multi-word queries like strain IDs.
>
> > Some notes I get from speed-reading the info in the first link; At least
> > somebody's classification system thinks this strain qualifies as
> > Biohazard Class/Level 2, which means you probably can't culture this
> > legally outside a Class 2 lab. Most lab strains of E.coli are Class 1,
> > meaning that most places have no laws requiring anything specific.
>
> > If this strain is Class 2 where you live, or if there's a good reason
> > why anyone thinks it's Class 2, perhaps you should look for another
> > strain. Given that a strain comes for free with the pGlo kit AFAIK, I'd
> > stick with that rather than this strain.
>
> > and getting a less hazardous culture; ideally you're looking for E.coli
> > K12 or something descended from K12. Nice cultures also include
> > "DH5-Alpha" or "DH10-Beta" (AKA "Top-10")
>
> > On 16/01/12 11:25, Mega wrote:
>
> > > I finally got non-pathogenic e.coli from a proffessor. it's the strain
> > > "dsm 1103". how can i find out if it has plasmids inside? on google i
> > > find some related articels but no gene map or description of the
> > > strain (i'm looking for a kind of datasheet if you will)
>
> > > best regards
>
> > > On 2 Jan., 14:24, Cathal Garvey <cathalgar...@gmail.com> wrote:
Avery louie
--A
--
Mega
Internship would be a great idea... Although in my country genetic
enginereering is quite restricted... So there may be not so much
companies...
""On the other hand, it does not seem
super dangerous to proceed. ""
knowing it's safe (when handled as sterile as possible...)
On 17 Jan., 17:45, Avery louie <inactiv...@gmail.com> wrote:
> Don't eat it. Sometimes there are restrictions on who you can share
> organisms with, specified in a materials transfer agreement, so it may be
> possible that they cannot give you anything else. I would try to get some
> kind of lab access via internship or research, it will let you learn a lot
> more with a lot less sketchyness. On the other hand, it does not seem
> super dangerous to proceed.
>
> --A
>
>
>
>
>
>
>
> On Mon, Jan 16, 2012 at 11:44 AM, Mega <masterstorm...@gmail.com> wrote:
> > Well, i asked my professor via e-mail about that and sent him those
> > > and getting a less hazardous culture; ideally you're looking for E.coli
> > > K12 or something descended from K12. Nice cultures also include
> > > "DH5-Alpha" or "DH10-Beta" (AKA "Top-10")
>
> > > On 16/01/12 11:25, Mega wrote:
>
> > > > I finally got non-pathogenic e.coli from a proffessor. it's the strain
> > > > "dsm 1103". how can i find out if it has plasmids inside? on google i
> > > > find some related articels but no gene map or description of the
> > > > strain (i'm looking for a kind of datasheet if you will)
>
> > > > best regards
>
> > > > On 2 Jan., 14:24, Cathal Garvey <cathalgar...@gmail.com> wrote:
> > > >> It's messy, so you're far better off buying your lysozyme