[iGEM] Jan, could u plz help on the experimental device?

[Lei Xing]

hi, Jan,

Would you mind to have a research on the apparatus we are going to use for the incubation of the E.Coli? We need two light source, red and blue with specific wavelengths. We also need a incubator to put all the E.Coli bacterials evenly spread in it. The incubator has to be transparent so the light is able to pass through.

Below is a page from Texas on building a light cannon, this might inspire on the design.

If you wanna have a feel on the normal bacterial incubator, plz contact Craig, he is an expert on this.

http://openwetware.org/wiki/LightCannon

 

 

Cheers!

--
Lei Xing

[c.ham...@gmail.com]

I don't see the point of using PCR. Well it's not so expensive -
probably around $60 to get the 2 primers so we can use PCR to confirm
the presence of our insert in the strain but there are other cheaper/
more efficient ways of doing that. As far as initially amplifying the
plasmid, we can just make the bacteria multiply it. Just thought
getting rid of some of the redundant steps will make the pitch look
better.

One thing with the incubator: bacteria usually sink to the bottom of
the flask even with vigorous shaking (growing on a shaker). I don't
know if it's possible to keep them in solution evenly. But do we need
evenness?

Craig

[Alisa]

I agree about the PCR. If we know the sequence we can diagnose using
digestions, and amplification can be done using transformation.
However, if we want to introduce a restriction site, we might need to
do this with PCR. I think this might be a different kit though. For
the other purposes, I do think that we should be able to manage
without PCR. Having said this, we might need to use PCR at some stage,
especially if we have trouble with transformations.

Craig, it is ture that bacteria sink. This is why we are trying to
make them float. We also do not want them to be shaken when we shine
light through them. If everything goes to plan, the bacteria should
remain in suspension for up to three days. I don't know about needing
them to be completely even, but we do need to stop them from sinking.

[Phillip Dodson]

I am all for cutting out steps.

The point of PCR was as insurance against a failure to transform with
the plasmid recovered off a letter or however it is sent. Plasmid will
be 6Kb approx. Don't know what success rates we can expect from this
procedure, but I know people who have had trouble. I am willing to be
guided by someone who does know the risks. I suggest that we design
and order $60 of primers as a backup unless this transformation method
is rated better than 97% probable success by someone who knows.
This will avoid delay in the event of trouble.

Regards Phil.

[Phillip Dodson]

Don't see that we necessarily need a transparent incubator, could use
a small light source.

[Craig]

If we are worried about success rates, I know more people who have had
trouble with PCR than transformation especially when using a PCR
machine that you've never used before. I have never had a failed
transformation (at least a few colonies of interest grow on the
plates). This is not transformation following ligation- which is when
some people seem to have trouble, but just straight transformation. We
should initially transform the plasmid into something like NM522
bacterial strains that have a high transformation frequency in order
to keep a stock. The oligos don't take very long to order: usually
2-3 working days. I'm not sure about 97%..very little is ever that
high in biology.

I talked to Alisa and now know what's going on with making the
bacteria float. But I don't use an incubator to grow my bacteria so I
am not even close to an expert on that. The one in our lab is huge,
for growing mammalian culture cells and we probably won't have access
to that kind of thing. It's easiest to ask Paul about whether we might
have access I'd think.

Craig