Re: [DIYbio] Re: Experiment with DNA and learn the techniques and concepts in a convenient and easy way

        As far as I know, the purpose of CaCl2 is no neutralize the negative charge of the DNA and on the bacterial membrane so that the plasmid is not repelled by the membrane. What we do is take a colony off a plate and then transfer them into two tubes, one containing CaCl2 and the plasmid and a control tube containing only CaCl2. Bacteria are resuspended using a loop and that's all you need before going into heat shock protocol. 

        For this particular experiment, we use a large amount of DNA to achieve transformation. Competent cells are obtained from fresh colonies of E coli growing in solid LB media, they are resuspended in CaCl2 so there is no need to wash LB off, since you are using solid media to propagate. So basically the transformation efficiency is not great and we compensate for that by using a lot of plasmid DNA to get the result. In our lab, when we want to clone something we've made by cutting and pasting DNA we need to prepare competent cells. Since we don't have a large centrifuge for bulk competent cell peparation - what we do is grow E coli untiil log phase then transfer 1.5 mL of culture to 1,5 microtubes, spin in a centrifuge at 6K RPM, add more culture into the same tube and repeat, and then resuspend in CaCl2, these are ready and competent and can be frozen at -20°C for later use, though I don't think they remain competent very long. 

Matías

A 

On Friday, March 9, 2012 1:59:05 AM UTC-3, Nathan McCorkle wrote:

Can you post the protocol? I know there have been people on the list
lately that have been trying to manage a GFP transformation without a
centrifuge (you didn't address how you get around this).

On Thu, Mar 8, 2012 at 7:20 PM, Matías Gutiérrez
<matiasgutierrezm@gmail.com> wrote:
> Also included, forgot to add it into the list. :) but not for propagation,
> the propagation is achieved by plating the bacteria in the solid LB media.
> The liquid LB is used for recovery after heat shock transformation.
>
> Basically the idea is that you'll have everything you need, we've tested
> these in classrooms for over 3 years with great results and have improved
> our kit each year, this would be the first version that is not meant for the
> classroom but for enthusiasts!
>
> We could also provide individual components in a convenient ammount so that
> ppl don't need to buy bulk like research labs usually do.
>
> Matías
>
>
> On Thursday, March 8, 2012 8:55:43 PM UTC-3, Nathan McCorkle wrote:
>>
>> Matias, what about liquid LB for propagation?
>>
>> On Thu, Mar 8, 2012 at 6:46 PM, Matías Gutiérrez
>> <matiasgutierrezm@gmail.com> wrote:
>> > Hey Guys,
>> >
>> >    First of all thanks to Edwin for your warm wishes :)
>> >
>> > Now regarding these kits, they basically include everything.
>> >
>> > For example the GFP cloning kit includes the following:
>> >
>> > plasmid encoding GFP and ampicilling resistance (GFP expression is
>> > induced
>> > by arabinose)
>> > bacterial stock culture (stab) that needs to be propagated before the
>> > experiment
>> > 2 plates containing propagation media (LB)
>> > plate containing selection media (LB + ampicillin)
>> > plate containing selection media and inducer (LB + ampicilling +
>> > arabinose)
>> > inoculations loops, microtubes, transfer pipettes, etc
>> > detailed protocol for the experiment
>> > Slide show with explanation of the protocol
>> > further reading material
>> >
>> > If we can work out a good distribuition network I'd really like to make
>> > this
>> > available for the community at a low cost (hopefully less than
>> > USD$30.00)
>> >
>> > Matías
>> >
>> > On Thursday, March 8, 2012 6:29:05 PM UTC-3, Avery wrote:
>> >>
>> >> You might just want to get pGREEN from carolina.  There is no need for
>> >> arabinose with that plasmid.
>> >>
>> >> --Avery
>> >>
>> >> On Thu, Mar 8, 2012 at 3:48 PM, Mega <masterstorm123@gmail.com> wrote:
>> >>>
>> >>> So you are still dependent on chemical companies   that may deny
>> >>> access.
>> >>>
>> >>> And if you (vacuum) destill the apple juice so the concentration gets
>> >>> higher? Is arab heat stable (say 100°C)?
>> >>>
>> >>> On 8 Mrz., 20:20, Derek <dere...@gmail.com> wrote:
>> >>> > Nope, no arabinose at the supermarket. It's found in low
>> >>> > concentrations in grape and apple juice, but I wasn't able to get
>> >>> > sufficient concentrations to enable GFP production.
>> >>> >
>> >>> > Best price I found, and nice people to deal with, are the folks at
>> >>> > Cascade Biochemicals,http://www.cascadebiochems.com/
>> >>> >
>> >>> > --Derek
>> >>> >
>> >>> > On Mar 8, 9:26 am, Mega <masterstorm...@gmail.com> wrote:
>> >>> >
>> >>> >
>> >>> >
>> >>> >
>> >>> >
>> >>> >
>> >>> >
>> >>> > > As for 2. GFP I want to mention, you need arabinose.
>> >>> >
>> >>> > > You can get it as DIY biologist, but not from the supermarket ( I
>> >>> > > think so, in fact never looked for arab there)
>> >>> >
>> >>> > > And you need a black light source.
>> >>> >
>> >>> > > There's a plasmid, pVIB, that is always 'on' and needs no special
>> >>> > > substrate. It may be more attracting to people if they see it
>> >>> > > glowing
>> >>> > > in the dark without UV-Lamps.
>> >>> > > That may only be true for me, at all. Though, I recommend thinking
>> >>> > > about this.
>> >>> >
>> >>> > > Best
>> >>>
>> >>> --
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>> >>
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>> --
>> Nathan McCorkle
>> Rochester Institute of Technology
>> College of Science, Biotechnology/Bioinformatics
>
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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics