Luciferases and Skype reunion
Hi Mariana,
What promoter and RBS are you using to express the luciferase? This
could be an issue.
If you are conducting an in vitro assay then you could try using
higher luciferin concentrations. We think that uptake of luciferin is
the limiting factor. Also we notice a burst of emission immediately
after adding the luciferin. One strategy would be to take a plate
into a dark room and add luciferin. Wait 10 minutes first to let your
eyes adapt.
We really never took any picture, as there was nothing to
see.
If you have a camera where you can set exposure time it is definitely
worth it, since 30s of exposure can capture a lot more than your eye.
We were wondering if you wanted to talk on Skype later? Might be
easier than email. We'll try to send you stuff soon.
Best
Theo and the rest of the team
Answer from UNAM_genomics_MEXICO team
Hi Theo and the rest of the team!
I assume that you're working also with the firefly luciferase then.
In the first trial that I made, I tried using a pBluescriptIIKS(+/-) and in
regards to the RBS, I took into account the experience of the registry with
this part and we synthesize a special primer to delete this bases:
"Note that sequencing shows an unexpected insertion of 6 bases, ACCACC,
after the scar between the ribosome binding site and the ATG of the
luciferase coding sequence; that is, the sequence reads ACTAGACCACCATG
rather than ACTAGATG. If this is correct, these 6 bases must be present in
luciferase BioBrick BBa_I712019, or else were introduced by a random event
during construction. This makes the RBS 12 bases from the ATG, somewhat
more than the optimum, but with a strong RBS (like J15001) should still
give about 25% of the expression level expected for optimum spacing in E.
coli (Vellanoweth & Rabinowitz, 1992, Molecular Microbiology 6,
1105-1114)."
Now, I'm planning to use a constitutive promoter (J23101) from the
registry.
About the skype meeting, my username is Mariana Ruiz Velasco, considering
the 6 hrs of difference, at which time do you think we can talk?
Bests,
Mariana Ruiz Velasco Leyva
Undergraduate Program on Genomic Sciences
6th generation
Express delivery instructions
Hi again Cambridge!
About the express delivery: please declare them as synthetic oligos and
avoid using the words "DNA", "plasmid", "bacterial", "biological" by all
means. I will also ask you to label the tubes by number and to avoid
descriptions, you can give me the details by mail later. Please tell me how
many tubes you are sending and the tracking number, as the customs protocol
requires this information.
The address is the one that I previously send to you and don't forget to
add the paragraph rewarding the non-hazardous, etc material.
My team and I thank you again for your willingness in helping us,
Bests.
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