Thermal Cycler Based Bacterial Trasformation

[Sebastian Cocioba]

Anyone have any luck doing transformations using their PCR machine?

This is my protocol done on a 0.5mL Biometra UNO with no heated lid:

One fresh colony of HB101 per tube in 250uL of cold CaCl2 solution (100mM) + 1ng of Plasmid. All initial dispensing done cold onto prechilled PCR block holding at 4C.

Chill at 4C for 30mins
Heat at 42C for 45sec
Chill at 4C for 2min
Add 250uL LB Broth
Heat at 37C for 50mins

I get nothing. I'm using a simple biobricked gfp construct with AmpR. Would a PCRs relatively slow ramp time screw up the shock part of heat shock? My ramp time is about 1.8C per sec.


Sent from my Windows Phone

[Sebastian Cocioba]

Water bath is down for cleaning. This was just a trial run. The allure
of semi-automated transformation tempted me to try this. What may be a
solution to the aeration issue? Less time? Dispense the transformation
reaction into pre warmed LB flasks and shake for a while with no
antibiotics? Thanks for the insight!

Sent from my Windows Phone From: Tom Knight
Sent: 8/16/2013 4:45 PM
To: Sebastian Cocioba
Cc: Tom Knight
Subject: Re: [DIYbio] Thermal Cycler Based Bacterial Trasformation
Your outgrowth at 37 is a problem with no aeration. Otherwise, this
will likely work. Did you do a control transformation with a normal
heat shock?

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[Avery louie]

Dude, that is an awesome idea.  I will have to try it.

I have considered a transformation machine, but it was far more mechanical.

--A

--

[Avery louie]

Actually, if you are using somethign with AmpR you should not do the recovery step, just plate immediately.  In my experience, recovery time leads to build up of b-lacatamase and satellite colonies.  Are you getting any growth at all?

--A

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[Avery louie]

Another control to run is just a non-transformed colony in the machine.  If there is no growth, something might be killing them (heated lid on?)

--A

[scoc...@gmail.com]

No heated lid, no growth. Its been 24hrs already. Plates are clean yet control run showed growth on LB no plasmid no amp. Gonna redo this again and with more design care as to actually control correctly. I'm looking for a good reference plasmid that can work reliably. Might want to actually integrate the net thermal change accounting for ramp time and see if it can even be called a shock...kinda fun.

 

Sent from Windows Mail

 

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[Aaron Vollrath]

i use this approach all the time.  mainly due to the fact i am extremely lazy.  i am not sure of the ramp time.  i don't think the thermocycler i have been using has an adjustable ramp time.  i can double-check if you are interested in what it is.  i usually transform 50uL of commercial competent cells and get colonies.  i have also done 10 uL of commercial competent cells w/ pGLO and had success.  i have not did a side-by-side comparison between using the thermocycler and the traditional approach to assess efficiency.  i get colonies, but have some issues when doing restriction free cloning, but that might be due to no or extremely low quantities of pcr amplified vector.

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[Nathan McCorkle]

I net the ramp time from ice bath to 42C bath is way more than any old/cheap thermal cyclers.

I know its probably simple math to find a decent transitions time to change 50uL water from 4C to 42C using the heat capacity of a few gallons of water in a heat bath at 42C.

Simon, can you mathify us?

--

[Avery louie]

@nathan, what do you mean?  I can probably mathify you.

--A

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[John Griessen]

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.

[Jeswin]

The time from 4C to 42C in the regular water bath method is
insignificant. 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.

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[Nathan McCorkle]

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.

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[jarlemag]

I think he meant that the typical ramp time is *insignificantly long*, while the ramp time is still a variable of *significant importance*.

-JP

[Sebastian Cocioba]

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

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[Koeng]

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

[Sebastian Cocioba]

Do you know the plasmid concentration and volume used? Cell strain? Sorry for the interrogation, the devil is in the details.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

---

From: Keoni Gandall
Sent: 8/20/2013 1:55 PM
To: Sebastian Cocioba
Subject: Re: [DIYbio] Thermal Cycler Based Bacterial Trasformation

I thawed the cells on ice, and once they where liquid I added the plasmid.

I had 2 tubes, one I transferred 100µl (all of it) to a 200µl pcr tube, and put it into my labs thermocycler. The protocol I set was 

10 minutes at 4C

45 seconds at 42C

5 minutes at 4C

The other tube I left out on ice for 15 minutes, which may be the reason for the lesser efficiency. After that I just did 42C in a water bath, 5 minutes on ice and then I plated both. (The plasmid was pUC19)

The manufacture says the ramp time is .1C to 5C a second, which I have to go with 5C since the machine is like almost new

On Aug 20, 2013, at 10:37 AM, Sebastian Cocioba <scoc...@gmail.com> wrote:

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

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[Keoni Gandall]

Its fine :)

I used .2ng of pUC19 (1µl) with the bacteria strain ss320, however I also tried it with ccdB resistant cells and got around the same results. The competent cells were in 100µl aliquots 

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[Nathan McCorkle]

> From: Keoni Gandall
> Sent: 8/20/2013 1:55 PM
> To: Sebastian Cocioba
> Subject: Re: [DIYbio] Thermal Cycler Based Bacterial Trasformation

> The manufacture says the ramp time is .1C to 5C a second, which I have to go
> with 5C since the machine is like almost new

I believe the lower ramp rate (0.1C/sec) is going from Hot to Cold,
while the faster ramp rate (5C/sec) is going from Cold to Hot. You
might re-read the manual to see if that's mentioned somewhere, but if
it's a peltier-based device, that's likely what these two numbers
mean.

[Sebastian Cocioba]

Ah, I thought it was a range due to the machines ability to set the
actual ramp rate. Didn't know the PCRs have different thermal rates but
now that I think about it it does make a ton of sense.

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

Plant Biotech R&D From: Nathan McCorkle
Sent: 8/20/2013 6:10 PM
To: diybio

Subject: Re: [DIYbio] Thermal Cycler Based Bacterial Trasformation
> From: Keoni Gandall
> Sent: 8/20/2013 1:55 PM
> To: Sebastian Cocioba
> Subject: Re: [DIYbio] Thermal Cycler Based Bacterial Trasformation
> The manufacture says the ramp time is .1C to 5C a second, which I have to go
> with 5C since the machine is like almost new

I believe the lower ramp rate (0.1C/sec) is going from Hot to Cold,
while the faster ramp rate (5C/sec) is going from Cold to Hot. You
might re-read the manual to see if that's mentioned somewhere, but if
it's a peltier-based device, that's likely what these two numbers
mean.

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[Sebastian Cocioba]

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

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[Keoni Gandall]

I believe that you don't have to wait to plate with ampicillin, but only with ampicillin. 

Sent from my iPhone

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[Cathal Garvey]

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.

> > 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: diy...@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

[Sebastian Cocioba]

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: diy...@googlegroups.com
Subject: Re: [DIYbio] Thermal Cycler Based Bacterial Trasformation

[Avery louie]

For lurkers/curious folks, here is what it looks like with a ~20 min incubation with a GFP plasmid:

http://tequals0.files.wordpress.com/2011/10/6060142073_8f633a418d_b.jpg
http://tequals0.files.wordpress.com/2011/10/6060132419_6f685da983_b.jpg

and without:

http://tequals0.files.wordpress.com/2012/03/igp5196.jpg

Notice in the first two, there are a lot of non-glowing (non-transformed) colonies, and the transformed and non transformed colonies are even mixed together.  In the last one, they are all glowing (transformed) and very round, which (theoretically) indicates that they grew up from a single cell.

--A

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