Re: [DIYbio] Thermal Cycler Based Bacterial Trasformation

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

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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

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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

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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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