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.
> >>> > >
> >>> > >
> >>> > > --
> >>> > > -- You received this message because you are subscribed to
> >>> > > the Google Groups DIYbio group. 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
> >>> > > https://groups.google.com/d/forum/diybio?hl=en Learn more at
> >>> > > www.diybio.org --- You received this message because you are
> >>> > > subscribed to the Google Groups "DIYbio" group.
> >>> > > To unsubscribe from this group and stop receiving emails from
> >>> > > it, send an email to diybio+un...@googlegroups.com.
> >>> > > To post to this group, send email to diy...@googlegroups.com.
> >>> > > Visit this group at http://groups.google.com/group/diybio.
> >>> > > To view this discussion on the web visit
> >>> > > https://groups.google.com/d/msgid/diybio/520F8A72.3090408%40industromatic.com.
> >>> > >
> >>> > > For more options, visit
> >>> > > https://groups.google.com/groups/opt_out.
> >>> >
> >>> > --
> >>> > -- You received this message because you are subscribed to the
> >>> > Google Groups DIYbio group. 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
> >>> > https://groups.google.com/d/forum/diybio?hl=en Learn more at
> >>> > www.diybio.org --- You received this message because you are
> >>> > subscribed to the Google Groups "DIYbio" group. To unsubscribe
> >>> > from this group and stop receiving emails from it, send an
> >>> > email to diybio+un...@googlegroups.com. To post to this group,
> >>> > send email to diy...@googlegroups.com. Visit this group at
> >>> > http://groups.google.com/group/diybio. To view this discussion
> >>> > on the web visit
> >>> > https://groups.google.com/d/msgid/diybio/CAAhF0RLr6Z8pELeRVdBskKXug%3Do8aTfXBpLF2rcvWeOK7Wuv%2Bw%40mail.gmail.com.
> >>> > For more options, visit
> >>> > https://groups.google.com/groups/opt_out.
> >>
> >> --
> >> -- You received this message because you are subscribed to the
> >> Google Groups DIYbio group. 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 https://groups.google.com/d/forum/diybio?hl=en Learn
> >> more at www.diybio.org --- You received this message because you
> >> are subscribed to the Google Groups "DIYbio" group. To unsubscribe
> >> from this group and stop receiving emails from it, send an email
> >> to diybio+un...@googlegroups.com. To post to this group, send
> >> email to diy...@googlegroups.com. Visit this group at
> >> http://groups.google.com/group/diybio. To view this discussion on
> >> the web visit
> >> https://groups.google.com/d/msgid/diybio/7e2fb25c-59ea-46fb-b525-3026a0694ced%40googlegroups.com.
> >> For more options, visit https://groups.google.com/groups/opt_out.
> >
> > --
> > -- 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 https://groups.google.com/d/forum/diybio?hl=en Learn
> > more at www.diybio.org --- You received this message because you
> > are subscribed to the Google Groups "DIYbio" group. To unsubscribe
> > from this group and stop receiving emails from it, send an email to
> > diybio+unsubscribe@googlegroups.com. To post to this group, send
> > email to diybio@googlegroups.com. Visit this group at
> > http://groups.google.com/group/diybio. To view this discussion on
> > the web visit
> > https://groups.google.com/d/msgid/diybio/f8019ed4-6341-42fa-b439-3aeb2c0ba07e%40googlegroups.com.
> > For more options, visit https://groups.google.com/groups/opt_out.
> > -- -- 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 https://groups.google.com/d/forum/diybio?hl=en Learn
> > more at www.diybio.org --- You received this message because you
> > are subscribed to a topic in the Google Groups "DIYbio" group. To
> > unsubscribe from this topic, visit
> > https://groups.google.com/d/topic/diybio/hRPBHhMkEcQ/unsubscribe.
> > To unsubscribe from this group and all its topics, send an email to
> > diybio+unsubscribe@googlegroups.com. To post to this group, send
> > email to diybio@googlegroups.com. Visit this group at
> > http://groups.google.com/group/diybio. To view this discussion on
> > the web visit
> > https://groups.google.com/d/msgid/diybio/-1887958013564660325%40unknownmsgid.
> > For more options, visit https://groups.google.com/groups/opt_out.
>

--
-- 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 https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+unsubscribe@googlegroups.com.
To post to this group, send email to diybio@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/-5020626025953520232%40unknownmsgid.
For more options, visit https://groups.google.com/groups/opt_out.