50 milliMolar is what I've seen used
On Tue, Feb 7, 2012 at 8:11 AM, Mega <masterstorm123@gmail.com> wrote:
> Again a question ;)
>
>
> Well, for the cacl2 transformation - whatconcentration will I need???
> I read 0.1 molar, 0.05molar, 0.07 molar??
>
> What suits best??
>
>
>
> On 6 Feb., 18:52, Mega <masterstorm...@gmail.com> wrote:
>> Ah, ok.
>>
>> I thought that the electricity can go through the cell wall anyway if
>> it's strong enough.
>> And exponential growth with thin cell walls is better for chemical
>> transformation.
>>
>> On 5 Feb., 21:37, Nathan McCorkle <nmz...@gmail.com> wrote:
>>
>>
>>
>>
>>
>>
>>
>> > I thought I stressed that in electroporation the cells do need to be
>> > mid-log/exponential phase. This is important because they're in a very
>> > healthy state nutrient-wise, and as Anselm pointed out their cell
>> > walls may be thinner (I've never heard that mentioned before, but I'm
>> > inclined to say it sounds reasonable)
>>
>> > Graphite sounds like decent electrodes, only one way to see if it works!
>>
>> > On Sun, Feb 5, 2012 at 4:10 AM, Mega <masterstorm...@gmail.com> wrote:
>> > > Well that sounds great...
>>
>> > > What I have planned so far:
>> > > Do a calcium chloride transformation and one PEG transformation and
>> > > see what works best under garage conditions.
>>
>> > > DIY electroporation also sounds great (do the cells in the
>> > > electroporator have to be in exponential growth?)
>>
>> > > For the electrodes I might try graphit, from an old battery (as my
>> > > former chemistry teacher recommended for making electrolysis - does
>> > > not corrode )
>>
>> > > On 5 Feb., 08:40, Nathan McCorkle <nmz...@gmail.com> wrote:
>> > >> On Sat, Feb 4, 2012 at 11:08 PM, Anselm Levskaya <levsk...@gmail.com> wrote:
>> > >> > Tips:
>>
>> > >> > Ampicillin - most definitely add it to the agar, you want a uniform
>> > >> > concentration of the stuff. Top spreading takes forever. Just add
>> > >> > the amp to your agar mix after it's cooled down to merely "hot" and
>> > >> > not boiling.
>>
>> > >> > Amp is decent at selecting from anywhere around 2ug/mL up to
>> > >> > 1000ug/mL, we typically use 50-100ug/mL in the lab. A light "dusting"
>> > >> > per plate by eye is generally enough. 100mg/L will work fine as a
>> > >> > recipe.
>>
>> > >> > Amp is very convenient in that you don't have to wait for the
>> > >> > resistance gene to be expressed before adding it. Since it attacks
>> > >> > cell wall growth and not translation, you can just add it. With
>> > >> > kanamycin, chloramphenicol, etc. you have to wait an hour.
>>
>> > >> > ---
>> > >> > Re competent cells: I agree that the TSS competency method is
>> > >> > probably the easiest to get to work in a home lab setting.
>> > >> > electrocompetent preps are even easier but you'd then need to
>> > >> > build/obtain a 2500V exponential wave shocker. (Highly recommend
>> > >> > electrocompetent methods if you want really high competency, i.e. if
>> > >> > you ever want to try a random library screen / selection.)
>>
>> > >> Mega this is pretty good info, you should try using a piezo electric
>> > >> sparker, commonly found in electronic ignition household butane
>> > >> lighters (the long grill lighters generally have them).
>>
>> > >> Read the two posts here with info compiled by Simon Quellen Field
>> > >> about how to use a piezo sparker and a potentiometer to adjust the
>> > >> voltage, which for E.coli is generally 18kV/cm (or 1.8kV if the
>> > >> electrodes in the cuvette are 0.1cm apart).
>>
>> > >>http://groups.google.com/group/diybio/browse_thread/thread/78979422c0...
>>
>> > >> I successfully electroporated mid-log phase (phase is important as
>> > >> Anselm said) E.coli with no preparation to the cells other than 2
>> > >> rinses with distilled and filtered water (filtered of ions such that
>> > >> the resistance of the water is about 18 Mega Ohms) (though distilled
>> > >> and sterile should be just fine).
>>
>> > >> I did use a commercial electroporator, and commercial cuvettes (you
>> > >> have to be careful of the metal used for the electrodes if you make
>> > >> your own reactor, use gold or platinum, because they are pretty
>> > >> non-reactive metals if they come into solution from the spark)
>>
>> > >> --
>> > >> Nathan McCorkle
>> > >> Rochester Institute of Technology
>> > >> College of Science, Biotechnology/Bioinformatics
>>
>> > > --
>> > > You received this message because you are subscribed to the Google Groups "DIYbio" group.
>> > > To post to this group, send email to diybio@googlegroups.com.
>> > > To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com.
>> > > For more options, visit this group athttp://groups.google.com/group/diybio?hl=en.
>>
>> > --
>> > Nathan McCorkle
>> > Rochester Institute of Technology
>> > College of Science, Biotechnology/Bioinformatics
>
> --
> You received this message because you are subscribed to the Google Groups "DIYbio" group.
> To post to this group, send email to diybio@googlegroups.com.
> To unsubscribe from this group, send email to diybio+unsubscribe@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
--
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To post to this group, send email to diybio@googlegroups.com.
To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com.
For more options, visit this group at http://groups.google.com/group/diybio?hl=en.
0 comments:
Post a Comment