Re: [DIYbio] Thermal Cycler Based Bacterial Trasformation

Thanks so much for the clarification. I completely forgot Amp doesn't
kill...just inhibits future growth.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC

Plant Biotech R&D From: Cathal Garvey
Sent: 8/22/2013 5:46 PM
To: diybio@googlegroups.com
Subject: Re: [DIYbio] Thermal Cycler Based Bacterial Trasformation

More generally; you don't have to wait with *bacteriostatic*
antibiotics, only with *bacteriocidal*. The former inhibit growth, the
latter kill the cells. So with the latter you need .5-1.5 hours
incubation to allow sufficient expression of resistance gene to protect
transformants, with the former you rather plate right away.

On Thu, 22 Aug 2013 11:53:09 -0700
Keoni Gandall <koeng101@gmail.com> wrote:

> I believe that you don't have to wait to plate with ampicillin, but
> only with ampicillin.
>
> Sent from my iPhone
>
> On Aug 22, 2013, at 10:41 AM, Sebastian Cocioba <scocioba@gmail.com>
> wrote:
>
> > So a modified version of my protocol worked:
> >
> > 100uL CaCl2 100mM
> > 1uL 1ng GFP Biobrick practice plasmid
> > 1 colony of 16hr HB101
> >
> > 10min 4C
> > 30sec 42C
> > 2min 4C
> > 30min 37C
> >
> > Plated on LB Amp IPTG overnight.
> >
> > I got colonies (about 30) but during a time trial experiment with 4
> > repeats of 15-45sec at 42C I got mixed results. I believe this is
> > due to the variation in colony pickup and dispersal in the
> > transformation solution. I'm going to do some backlogged leg work
> > and find the CFU/mL of my strain and grow conditions and only use
> > standardized liquid suspensions. Using a pipette tip to pick and
> > place colonies is just too variable. Do you guys think the
> > outgrowth at 37c for 30min would actually be detrimental. Dr.
> > Knight stated earlier that this may be an issue especially since
> > this new protocol does not add any SOC or LB Broth during
> > outgrowth. I mean the bacteria would need some time to express the
> > AmpR before their next mitotic event....but again that's just
> > theory. Someone said they plate directly after cold recovery and
> > skip outgrowth. I guess by the time some cells, which started their
> > cycle upon plating, multiply the AmpR would already be in effect?
> >
> > Sebastian S. Cocioba
> > CEO & Founder
> > New York Botanics, LLC
> > Plant Biotech R&D
> > From: Sebastian Cocioba
> > Sent: 8/20/2013 1:37 PM
> > To: Koeng
> > Subject: RE: Fwd: [DIYbio] Thermal Cycler Based Bacterial
> > Trasformation
> >
> > What was your protocol exactly? Do u know your machine's ramp time?
> > Just want to compare notes.
> >
> > Sent from my Windows Phone
> > From: Koeng
> > Sent: 8/20/2013 1:36 PM
> > To: diybio@googlegroups.com
> > Subject: Re: Fwd: [DIYbio] Thermal Cycler Based Bacterial
> > Trasformation
> >
> > I just tried it, I got 2-3 times better results with the
> > thermocycler...
> >
> > Then again i might have left the cells on ice too long ect, just
> > saying what i got
> >
> > -Koeng
> >
> > On Sunday, August 18, 2013 5:54:37 AM UTC-7, Sebastian wrote:
> >>
> >> Given the average used PCR machine has a ramp time of about 2
> >> degrees per second, it would be a very significant variable, no?
> >> The thin walled pcr tubes are made to transfer heat as quickly as
> >> possible. A swing from 4c to 42c at 2c/sec would still be 19
> >> seconds. I've managed to get colonies using traditional heat shock
> >> with only 10sec exposure. Also has anyone ever proven
> >> experimentally that a membrane pore formation is what is actually
> >> occurring? Like freeze fracture electron microscopy or something
> >> to depict the pores? Just curious.
> >>
> >> Sent from my Windows Phone
> >> From: jarlemag
> >> Sent: 8/18/2013 5:54 AM
> >> To: diy...@googlegroups.com
> >> Subject: Re: Fwd: [DIYbio] Thermal Cycler Based Bacterial
> >> Trasformation
> >>
> >> I think he meant that the typical ramp time is *insignificantly
> >> long*, while the ramp time is still a variable of *significant
> >> importance*.
> >>
> >> -JP
> >>
> >> kl. 06:41:23 UTC+2 søndag 18. august 2013 skrev Nathan McCorkle
> >> følgende:
> >>>
> >>>
> >>> On Aug 17, 2013 12:53 PM, "Jeswin" <phill...@gmail.com> wrote:
> >>> >
> >>> > The time from 4C to 42C in the regular water bath method is
> >>> > insignificant.
> >>>
> >>> Your comment later about ramp rate means it /is/ significant.
> >>>
> >>> Some think the thermal shock creates a pressure wave.
> >>>
> >>> The NEB competent cells I work with are only held at
> >>> > 42C for 30s. My guess is that the ramp time on a PCR machine is
> >>> > significant compared with the time held at 42C. From what I
> >>> > recall of the theory, the sudden shock of 42C opens up the
> >>> > pores allowing DNA to enter. Slowly ramping probably negates
> >>> > the heat-shock effect.
> >>> >
> >>> > On Sat, Aug 17, 2013 at 10:36 AM, John Griessen
> >>> > <jo...@industromatic.com> wrote:
> >>> > > On 08/17/2013 12:17 AM, Avery louie wrote:
> >>> > >>
> >>> > >> @nathan, what do you mean?  I can probably mathify you.
> >>> > >
> >>> > >
> >>> > > It all depends on the heat conductivity of the vial.
> >>> > >
> >>> > > Thin vials with agitation --> fast heat ramps.
> >>> > >
> >>> > >
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