Re: [DIYbio] Free plasmids for amateur biologists.

[Nathan McCorkle]

Nice find! They're based in the U.S. but also have a location in Germany too.

On Sat, Jun 30, 2012 at 7:03 AM, Александр Дориф <dor...@gmail.com> wrote:
> Hello! I have found the way to get some free plasmids with map and work
> protocol. The Aldevron company distributes free samples of pRc/CMV-Hbs(S)
> and pCMVHB-S2.S plasmids. Both are based on pBlueScript plasmid and contain
> Ampicilline resistance gene. Each sample contains 2-4 ug of plasmid DNA.
> Samples are delivered via postage service. Link to order
> samples: http://www.aldevron.com/products/dnas/plasmids/
>
> I hope it'll be useful for somebody.
>
> P.S.: sorry for my bad English.)
>
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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Dorif]

I know.) But in Europe are located antibody producing facilities(Aldevron Freiburg, former Genovac), DNA production is located in Fargo, North Dacota(from where i received my plasmids).

And I have two questions: a) from where can I obtain E.coli strain, suitable for transformation with these plasmids(of course, I have wild strains of E.coli and plasmids are in fridge(so, I can wait a little), but...), b) With what I can replace TrisEDTA beffer, recommended for plasmid extraction from samples(Currently I have no Good's buffers at all, but I have wide range of inorganic compounds, suitable for PBS, potassium-phosphate buffer, water, acetate buffer, oxalate buffer)?

воскресенье, 1 июля 2012 г., 22:55:46 UTC+3 пользователь Nathan McCorkle написал:

Nice find! They're based in the U.S. but also have a location in Germany too.

On Sat, Jun 30, 2012 at 7:03 AM, Александр Дориф <dor...@gmail.com> wrote:
> Hello! I have found the way to get some free plasmids with map and work
> protocol. The Aldevron company distributes free samples of pRc/CMV-Hbs(S)
> and pCMVHB-S2.S plasmids. Both are based on pBlueScript plasmid and contain
> Ampicilline resistance gene. Each sample contains 2-4 ug of plasmid DNA.
> Samples are delivered via postage service. Link to order
> samples: http://www.aldevron.com/products/dnas/plasmids/
>
> I hope it'll be useful for somebody.
>
> P.S.: sorry for my bad English.)
>
> --
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To view this discussion on the web visit
> https://groups.google.com/d/msg/diybio/-/R1F7yg1algYJ.
> To post to this group, send email to diy...@googlegroups.com.
> To unsubscribe from this group, send email to

> diybio+unsubscribe@googlegroups.com.

[Dan Wright]

You should not work with wild strains of ecoli. It is way too dangerous. It is best to wait until you can get the safe strain from a local university or supplier.

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[Dorif]

I know, what it's too dangerous. So, I can go to the faculties of Biology or Medicine and they can give me known strains of E.coli? Ok, i'll do it. But what about buffers?

понедельник, 2 июля 2012 г., 10:09:10 UTC+3 пользователь Roninlaw написал:

[Mega]

Great discovery!!!

Did I understand the mechanism of those plasmids correctly?

You transform bacteria with the pBluescript (derivate). And select for it.

Then, all you have to do is put a helper phage on the petri dish.

The sequence is then inserted. 

(?)

[Dorif]

Even better: you can use thermal shock transformation and no phage is needed. These plasmids carry AmpR operon, so, E.coli with one of these plasmids will be Ampicilline-resistent.

понедельник, 2 июля 2012 г., 13:42:47 UTC+3 пользователь Mega написал:

[shamrock]

Maybe I'm misunderstanding something here but, I would be a little circumspect about using these plasmids in your home biology experiments. The plasmids encode surface proteins from the Hepatitis B virus. Since the genes are under control of a mammalian promoter (CMV) you are unlikely to get expression of the protein in E. coli but why mess with it. I think you should avoid working with pathogens or parts of pathogens until you are sure of what your doing, have a good reason for doing so, and have assessed the risks.  

 

 If all your interested in is learning how to do transformations then use something like pUC-19 or pGFP, or something else inocuous. I'd be happy to send you some (if I can). Contact me off list and let me know where you live and I'll see if I can send some plasmid without jumping through a bunch of red tape.

 

Actually I'll make this offer to anybody on the list-I would rather see people who want to learn this technology work with something that is safe rather then take a chance with something that could cause some problems and could raise some eyebrows if it was found that you are working with Hepatitis genes. It's a perception issue.

[Cathal Garvey]

You can suspend the plasmids in deionised water safely, but it won't be
nearly as stable as TE buffer. It's fine if you suspend it right before
transformation or whatever other use you have, but TE is best for long
term storage.

If you have plasmid left after suspending in water, put it right in the
freezer, and try to minimise freeze/thaw cycles.

You can get tris and EDTA on ebay from a seller called "scuddlebutt3", I
think. If not, I got my tris originally from these guys:
http://www.atomscientific.com/products.php?category_range=buffers#T

The EDTA isn't essential provided you find another way to buffer the
tris to pH ~8.5, but it's recommended for its ability to chelate metal
ions and protect DNA. EDTA should be easy to find on ebay, it's used for
making soaps etc. and as a water softener.

On 02/07/12 07:49, Dorif wrote:
> I know.) But in Europe are located antibody producing facilities(Aldevron
> Freiburg, former Genovac), DNA production is located in Fargo, North
> Dacota(from where i received my plasmids).
>
> And I have two questions: a) from where can I obtain E.coli strain,
> suitable for transformation with these plasmids(of course, I have wild
> strains of E.coli and plasmids are in fridge(so, I can wait a little),
> but...), b) With what I can replace TrisEDTA beffer, recommended for
> plasmid extraction from samples(Currently I have no Good's buffers at all,
> but I have wide range of inorganic compounds, suitable for PBS,
> potassium-phosphate buffer, water, acetate buffer, oxalate buffer)?
>

> О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫, 1 О©╫О©╫О©╫О©╫ 2012 О©╫., 22:55:46 UTC+3 О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫ Nathan McCorkle
> О©╫О©╫О©╫О©╫О©╫О©╫О©╫:

>>
>> Nice find! They're based in the U.S. but also have a location in Germany
>> too.
>>

>> On Sat, Jun 30, 2012 at 7:03 AM, О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫О©╫ О©╫О©╫О©╫О©╫О©╫ <dor...@gmail.com>

>> wrote:
>>> Hello! I have found the way to get some free plasmids with map and work
>>> protocol. The Aldevron company distributes free samples
>> of pRc/CMV-Hbs(S)
>>> and pCMVHB-S2.S plasmids. Both are based on pBlueScript plasmid and
>> contain
>>> Ampicilline resistance gene. Each sample contains 2-4 ug of plasmid DNA.
>>> Samples are delivered via postage service. Link to order
>>> samples: http://www.aldevron.com/products/dnas/plasmids/
>>>
>>> I hope it'll be useful for somebody.
>>>
>>> P.S.: sorry for my bad English.)
>>>
>>> --
>>> You received this message because you are subscribed to the Google
>> Groups
>>> "DIYbio" group.
>>> To view this discussion on the web visit
>>> https://groups.google.com/d/msg/diybio/-/R1F7yg1algYJ.
>>> To post to this group, send email to diy...@googlegroups.com.
>>> To unsubscribe from this group, send email to

>>> diybio+un...@googlegroups.com.

>>> For more options, visit this group at
>>> http://groups.google.com/group/diybio?hl=en.
>>
>>
>>
>> --
>> Nathan McCorkle
>> Rochester Institute of Technology
>> College of Science, Biotechnology/Bioinformatics
>>
>

--
www.indiebiotech.com
twitter.com/onetruecathal
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PGP Public Key: http://bit.ly/CathalGKey

[Avery louie]

I also posted here a while ago, but I will send you gfp plasmids for free, with various buffers and reagents needed for the transformation.

--A

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[Mega]

Oh, really... You're right, it encodes a hepatitis surface protein.

At first I just read "pBluescript for free" and was thrilled about it...

(But given the plasmid map and that the gene of interest is within the needed restriction sites, you could just cut it out and kill it in the pressure cooker. )

Am Samstag, 30. Juni 2012 13:03:47 UTC+2 schrieb Dorif:

[Nathan McCorkle]

Those plasmid maps really, really suck... it's great to see the
restriction sites, but for anyone here that may ever label a plasmid
map, please add the intentional features as well (ori, selection
markers, MCS, promoter)

Shamrock is right that this is meant for mammalian cell line use, so
while it may replicate in E.coli, it probably isn't much fun. That
said, I don't think there's any problem with a protein, or fragment
thereof, being derived from Hepatitis B... it's not the whole virus,
so it can't do too much other than maybe stimulate an immune response
(which may or may not be severe)

On Sat, Jun 30, 2012 at 7:03 AM, Александр Дориф <dor...@gmail.com> wrote:
> Hello! I have found the way to get some free plasmids with map and work
> protocol. The Aldevron company distributes free samples of pRc/CMV-Hbs(S)
> and pCMVHB-S2.S plasmids. Both are based on pBlueScript plasmid and contain
> Ampicilline resistance gene. Each sample contains 2-4 ug of plasmid DNA.
> Samples are delivered via postage service. Link to order
> samples: http://www.aldevron.com/products/dnas/plasmids/
>
> I hope it'll be useful for somebody.
>
> P.S.: sorry for my bad English.)
>

> --
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To view this discussion on the web visit

> https://groups.google.com/d/msg/diybio/-/R1F7yg1algYJ.

> To post to this group, send email to diy...@googlegroups.com.
> To unsubscribe from this group, send email to
> diybio+un...@googlegroups.com.
> For more options, visit this group at
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[Cathal Garvey]

Kill what in the pressure cooker? DNA will happily survive pressure
cooker temperatures, although you might deaminate a few thymines here
and there.

The risks of handling this DNA aren't large, unless you snort it
(plasmid DNA can apparently be delivered in tiny amounts to nasal cells
in this manner, go figure). The antigen is probably the one used for
vaccination, but you don't want to express that in your own cells, for
fear of generating a more generalised autoimmune response.

As to putting it into other cells, depending on your region this may be
illegal: Hepatitis is probably on the US select organisms list, for
example, which puts work with Hepatitis DNA squarely in the "possibility
of having house stormed by angry unsympathetic soldiers" bracket.

In the EU, as with anything else you'd need a risk assessment of the
gene cassette and permission from your local GMO-regulating "competent
authority": if you can convince them that the protein doesn't carry the
infectious risks of the organism of origin, they might allow it as a
Class 1 Containment GMM, meaning you can work with it in a licensed
Class 1 lab. Don't even daydream of getting Heptatitis DNA deregulated:
it's already nigh-impossible to get them to deregulate things like GFP
without *all* the money.

[Dorif]

Much more better maps are shipped with free samples - with restriction sites, with genes location. And yes as it says Mega, Hepatitis B genes can be cutted off with restriction enzymes(If I had them) and plasmide can be used in other way - Aldevron writes it in papers, shipped with plasmids.

понедельник, 2 июля 2012 г., 18:22:37 UTC+3 пользователь Nathan McCorkle написал:

> diybio+unsubscribe@googlegroups.com.

[Dorif]

May be cooker won't destroy the DNA, but hot alkaline solution, used for hydrolysis of some organic compounds(I study at the Faculty of Chemistry, and my profile is Organic Chemistry, we use hydrolysis reactions to remove protector groups) - will.) And, fortunately, Moldova isn't a part of European Union at now, so, I don't need any permissions(I've read our laws).)

понедельник, 2 июля 2012 г., 18:31:00 UTC+3 пользователь Cathal написал:

[shamrock]

0.1 M HCl will do the trick as well and lots nicer to work with then hot alkali.

[Dorif]

But alkali are odorless.) And if you want to use acis - let it be diluted sulfuric acid - it hasn't terrible odor like HCl and if hot sulfuric acid drops to your skin - chemical scorch will be easier, then in case of alkali solution. It is simply my habit to use alkali for hydrolysis

понедельник, 2 июля 2012 г., 19:48:23 UTC+3 пользователь shamrock написал:

[Dorif]

It definitevely won't be a crime, if I'll publish here a link to the map, what I received with the plasmids.) http://img341.imageshack.us/img341/512/mappwa.jpg

суббота, 30 июня 2012 г., 14:03:47 UTC+3 пользователь Dorif написал:

[Dorif]

So, at map all of us can see, what if we want want to removeHepatitis B genes from the plasmid, we can cut them off with help of XhoI, BamHI, KpnI and BssHII restriction enzymes. After this, instead of Hepatitis B genes we can insert something useful, replicate plasmid in E. coli, re-extract plasmid from them and implant needed gene to the target mammalian. Or even more: we cat cut off CMV promoter with NruI restriction enzyme and insert, for example GFP operon or something like that, to be expressed directly in bacteria.

вторник, 3 июля 2012 г., 17:28:24 UTC+3 пользователь Dorif написал:

[Dorif]

Free cloning vector: http://www.psparkcloning.com/web/index/index Just add you gene.)

[Dorif]

Yes, even on this map isn't visible T7 promoter region in this plasmid. I was wrong what these plasmids are pBluScript derivatives - they actually are pCDNA3 plasmid derivatives. So, I analysed the plasmids sequence with help of UniPro Ugene and I've found, what between CMV promoter and genes coding HBsAg proteins is located T7 promoter, like in pCDNA3.

вторник, 3 июля 2012 г., 17:28:24 UTC+3 пользователь Dorif написал:

It definitevely won't be a crime, if I'll publish here a link to the map, what I received with the plasmids.) http://img341.imageshack.us/img341/512/mappwa.jpg