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.
On Fri, Aug 16, 2013 at 1:56 PM, <scocioba@gmail.com> wrote:
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 MailAnother 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
On Fri, Aug 16, 2013 at 4:51 PM, Avery louie <inactive.e@gmail.com> wrote:
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
On Fri, Aug 16, 2013 at 4:49 PM, Sebastian Cocioba <scocioba@gmail.com> wrote:
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?
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/-7058134866652058439%40unknownmsgid.
On Aug 16, 2013, at 4:41 PM, Sebastian Cocioba wrote:
> 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
>
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