RE: [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.

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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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