Hi,
I’ve received a number of concurring suggestions. Some have requested more
detail about the experiments. Here are the details.
1. Cells: BL21(DE3)
2. Plasmid: pET28a (T7 promoter)
3. Media: TB
4. Protein: cytoplasmic
5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce with
Thank you for your comments everyone.
The CCP4BB is a wonderful resource and it has answered several questions
that have been bothering me for years!
Tristran has given us the correct conclusion as well as the important
facts: the capacity of oil for holding O2 is high, but the diffusion rate
is
Hi Reza,
A few month ago I had a the exact same problem and we checked everything
we could think of but without any improvement. Finally we were able to
solve the problem only by subcloning the ORF into another plasmid (pET9a
or pET29b). The big difference being the His tag position (C-ter or
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Dear Smith,
the starting position for the first baton is the centre of your
screen, i.e. the pink box that usually resides there.
I don't understand your second remark. If it is connected to the
numbering, you can renumber the chain once it is
Hi,
Cultures are being properly cooled prior to induction (water bath supplemented
with ice).
Reza
Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
New York, NY 10031
http://www.khayatlab.org/
212-650-6070
From: lieh low [mailto:liehy...@gmail.com]
Sent:
Dear all,
at the ECM-29 in Rovinj, Croatia, there will be a microsymposium with the title
Structure solution on the fly - parallel data collection and structure
analysis
The description of the MS reads like this:
Recent developments at synchrotron beamlines are enabling users to collect
very
Reza,
someone might have mentioned this, it takes longer time to cool down larger
culture, we turn down the temp of the shaker when OD is about 0.4. For some
protocol, we even use ice to cool the flask down before induction. You
might also want to consider a lower induction temp, like 16degC.
Dear all,
sorry for off topic discussion.
I am having trouble transferring protein onto PVDF membrane for N-terminal
sequencing. When I used Tris-Glycine-Methanol in transfer buffer I got good
blot, but when I used CAPS buffer (pH 11 set with 2N NaOH and 10% methanol)
I did not get any blot. I
Dear CCP4ers,
Sorry for this non-structural biology related jobs announcements, but this will
be very helpful if you could spread this message to who might be interested.
Two postdoctoral researcher positions are available in the Department of
Medical Biochemistry and Biophysics at Karolinska
Dear Colleagues,
we are pleased to announce the third CCP4 structure solution school at the
Okinawa Institute of Science and Technology (OIST), Okinawa. All details can be
found at http://www.ccp4.ac.uk/schools/OIST-2015
Title:
CCP4 school: From data processing to structure refinement and
Hi Reza,
In addition to the many useful suggestions already made, I would suggest
lowering the final concentrations of IPTG. In many cases, 1mM IPTG
interferes with expression levels and/or solubility. This suggestion does
not address your concern for why things become ugly in going from 3mL to
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