[ccp4bb] Job Openings - Small Molecule Crystallographer (MicroED) and Scientific Programmer

[Jessica Bruhn]

Hi everyone,

My group is looking to hire two new people to further expand our small
molecule MicroED capabilities here at NanoImaging Services in San Diego,
CA. This is an exciting opportunity to help grow and shape our newest
service division; and bring impactful crystal structures to chemists
working in the pharmaceutical space.

Plus, we received an SBIR grant from the NIH to further develop our MicroED
pipeline, so these positions will have a significant R component. Come be
part of an innovative team that is taking cutting edge technology and
making it accessible to groups all across industry.

*Scientist I/II – Small Molecule MicroED Crystallographer (Full time – San
Diego - CA) *

https://www.nanoimagingservices.com/about-us/careers#Scientist_I-II_Small_Molecule_MicroED

*Scientific Programmer I (Full time – San Diego - CA)*

https://www.nanoimagingservices.com/about-us/careers#Scientific_Programmer_I

Inquiries can be sent to recruit...@nimgs.com or me directly.

Kind regards,
Jessica

-- 
Jessica Bruhn, Ph.D
Principal Scientist
NanoImaging Services, Inc.
4940 Carroll Canyon Road, Suite 115
San Diego, CA 92121
Phone #: (888) 675-8261
www.nanoimagingservices.com



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Re: [ccp4bb] Eukaryotic protein expression

[David Briggs]

Hi Dhiraj,

For transient expression in HEK293 cells in suspension, I generally use 
pcDNA3.1 which also uses a CMV promoter. Yields range from nothing to 50mg/l, 
depending entirely on what it is you're making.

I would suggest that maybe the promoter is not at fault, and perhaps the 
product is. Tweaking construct boundaries and optimizing transfection 
conditions may help.

Good luck,

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Srivastava, 
Dhiraj 
Sent: Thursday, June 24, 2021 9:42:32 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Eukaryotic protein expression


External Sender: Use caution.

Hi all
I am trying to express my protein in HEK293 cells for crystallization 
purpose but getting poor expression. I am using cmv promoter. Which 
promoter/vector people use to get good expression in eukaryotic expression 
system?

Thank you

Dhiraj



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The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



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Re: [ccp4bb] Eukaryotic protein expression

[Tomas Malinauskas]

Hi Dhiraj,

pHLsec vector for transient and its variants for lentivirus-based
expression (PMIDs 17001101 and 30455477, respectively).

Best wishes,
Tomas

On Thu, Jun 24, 2021 at 9:42 PM Srivastava, Dhiraj
 wrote:
>
> Hi all
> I am trying to express my protein in HEK293 cells for crystallization 
> purpose but getting poor expression. I am using cmv promoter. Which 
> promoter/vector people use to get good expression in eukaryotic expression 
> system?
>
> Thank you
>
> Dhiraj
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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[ccp4bb] Eukaryotic protein expression

[Srivastava, Dhiraj]

Hi all
I am trying to express my protein in HEK293 cells for crystallization 
purpose but getting poor expression. I am using cmv promoter. Which 
promoter/vector people use to get good expression in eukaryotic expression 
system?

Thank you

Dhiraj



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[ccp4bb] Macromolecular Crystallography - CSHL Course

[Paul Adams]

Dear Colleagues,

The July 16th deadline for applications to the CSHL X-ray Methods in Structural 
Biology Course to be held later this year, October 4 through October 19, 2021 
is rapidly approaching.

Please see the attached announcement, and the official course web site is here:

https://meetings.cshl.edu/courses.aspx?course=C-CRYS=21

please pass this on to folks who might be interested and who would benefit. The 
course is designed for researchers who are either new to, or who wish to 
increase their in-depth knowledge of, macromolecular crystallography. 

We anticipate holding the course in person as long as the pandemic situation 
continues to improve. Appropriate COVID-19 precautions will be followed to 
ensure the safety of all participants. Everyone will be required to provide 
documentary proof of a prior FDA or EMA approved vaccine. 

This immersive course is an outstanding place to learn both the theoretical and 
practical aspects of Macromolecular Crystallography because of the extensive 
lectures from world-renowned teachers, which are combined with hands-on 
experiments. This 2021 course will be lead by Jim Pflugrath (Rigaku, ret.), 
Tassos Perrakis (NKI), Paul Adams (LBL), Janet Newman (CSIRO), Tom Peat (CSIRO) 
and an All-Star cast of lecturers. Attendees can expect to participate in a 
course that is unparalleled in the world, with experts each devoting several 
days to teaching the fundamentals, theory and practical considerations of 
crystallographic structure solution.

We expect to have the participants crystallize several proteins and determine 
their structures all in about two weeks. Students may also work on their own 
projects, but not exclusively. They will also become well-versed in the theory 
of X-diffraction and crystal structure determination while having lots of fun, 
but not much sleep. There will be data collection at state-of-the-art beamlines 
at NSLS-II.

The course is limited to 16 participants due to the very hands-on nature of the 
experiments and the intimate seminar room and laboratory settings.  Please 
check the above web link for more details. In particular, please note the 
information about fellowships, scholarships, and stipends that are available. 
It is often possible for applicants to receive some level of support to help 
offset the course fee.

This course is supported with funds provided by the National Institute of 
General Medical Sciences for which we are extremely grateful. Also there are 
stipends available from the Leona M. and Harry B. Helmsley Charitable Trust and 
the Howard Hughes Medical Institute to help offset the cost of tuition. 
Scholarship support is provided by Regeneron Pharmaceuticals.

If anyone has any questions, please send me e-mail, I will be happy to answer 
any queries.

Thanks for spreading the word, Janet, Tom, Tassos, Paul, and Jim

> Begin forwarded message:
> 
> From: Cold Spring Harbor Laboratory Meetings & Courses 
> Subject: Macromolecular Crystallography - CSHL Course
> Date: June 24, 2021 at 5:20:20 AM PDT
> To: pdad...@lbl.gov
> Reply-To: meetm...@cshl.edu
> 
> 
>  
> 
>  
> 
> Macromolecular Crystallography 
> 
> October 4 - 19, 2021
> Application & Materials Deadline: July 16
> We anticipate that the vaccine situation may sufficiently improve to allow us 
> to offer this course safely in-person.
> 
> All participants will be required to provide documentary proof of a prior FDA 
> or EMA approved vaccine.  
> Instructors
> Janet Newman, CSIRO, Australia (likely remote)
> James Pflugrath, Rigaku Americas Corporation (ret.), Texas
> Anastassis Perrakis, Netherlands Cancer Institute
> Paul Adams, Lawrence Berkeley National Laboratory
> Co-Instructor:
> Tom Peat, CSIRO, Australia(likely remote)
> X-ray crystallography has been the cornerstone of structural biology for half 
> a century, and remains the technique of choice for atomic resolution 
> understanding of macromolecules and for structure guided drug discovery. This 

Re: [ccp4bb] missing covalent bond between asparagine and N-acetylglucosamine using coot carbohydrate module and phenix.refine

[Hinrichs, Winfried]

Dear Jiang, 



to draw a line or a dotted line is a question of graphics and
definitions of the specific software and has nothing to do with the
specific nature of a chemical bond. 
"link" defines in your case only an additional chemical bond that is
not defined for the common polypeptide chain. 



best, 

Winfried Hinrichs

--
Dr. rer. nat. Winfried Hinrichs
Professor and Chair of Biochemistry, Emeritus

University of Greifswald
Institute for Biochemistry
Felix-Hausdorff-Str. 4
D-17487 Greifswald, Germany

E-mail winfried.hinri...@uni-greifswald.de

http://orcid.org/-0002-0435-4565

--




Am Donnerstag, den 24-06-2021 um 13:40 schrieb jiang:


Hello guys,     I am a little bit confused. Since the
polysaccharide is attached to asparagine via covalent bond. I am not
sure what “link” means anyway.  Does it mean some other chemical
bonds? Or not chemical bond at all.
      As you can see from the book link below, the nitrogen atom
 from the N-glycosylation site has covalent bond with only one
hydrogen atom. The bond is referred to as glycosidic bond.
 https://www.ncbi.nlm.nih.gov/books/NBK22521/
     
Thank you,
Best,
Jiang
Sent from my iPhone



On Jun 24, 2021, at 12:35 AM, Folmer Fredslund  wrote:






Dear Jiang


As Paul Emsley was already kind to point out:


Coot draws solid lines for bonds between neighbouring monomers in a
polymer.
The bond between NAG and ASN is not such a bond - it is a link.
Links are drawn as dashed lines in Coot.



So, a dashed line is simply how such a bond is represented in Coot.
It's not a bug in the software.


I'm not quite sure why you want the bond to be represented with a
solid line instead of a dashed line. 
It makes no difference whatsoever on the validity of the model.


Best wishes
Folmer Fredslund










ons. 23. jun. 2021 01.23 skrev Jiang Xu :



Hello David,    I used the "Make Link" under the "Modeling..." tab
to create the bond. It worked, but the bond displayed is still a
dashed line, not a solid line as shown below. I still don't know how
to use phenix to add a solid bond between the two atoms.  So, is it a
bug in the software or I didn't do it correctly? 


Thank you,
Best,
Jiang


On Tue, Jun 22, 2021 at 3:48 PM Jiang Xu  wrote:



Hello David,    I tried to rebuild the polysaccharide chain using
the N-link add NAG, NAG, BMA method, saved the model and ran
phenix.refine without checking the "automatically add hydrogens to the
model". I also double checked to make sure that the "Link
carbohydrates to protein and other carbohydrates" within the
"Automatic Linking Options" was checked. The "Carbohydrate bond
cutoff" is the set to the default value of 1.99, which I didn't
change.  However, after running phenix.refine, the bond disappeared
again. As shown below, on the left is the input model, on the right is
the refined output. You can see that the bond is gone and the distance
between C1 of NAG and the Nitrogen atom of the amine group is 1.42 A.
So Could anything else cause the problem?




Thank you,
Best,
Jiang


On Mon, Jun 21, 2021 at 11:11 PM David Briggs  wrote:



   Hi Jiang Xu,

 

 (Phenixbb added as I suspect this is Phenix not coot that has caused
your issue)


 Did you check the "add hydrogens" button in Phenix.refine? Sometimes
this can erroneously add a second H to the ND of glycosylated Asn
residues. (I assume this is because bond distances in the input PDB
file fall outside Phenix's definitions of an Asn-NAG link).
 

 If the second H is added, this breaks the refinement of the Asn-NAG
bond.


 My work around is to run "Ready Set" to add hydrogens as a separate
job, and then manually inspect and correct the Asn residues as
necessary before running Phenix.refine and _not_ checking the "add
hydrogens" box.
 

 I hope this fixes your problem.
 

 Good luck,
 

 Dave
 

   --

 Dr David C. Briggs

 Senior Laboratory Research Scientist

 Signalling and Structural Biology Lab

 The Francis Crick Institute

 London, UK

 ==

 about.me/david_briggs [1]



-
 From: CCP4 bulletin board  on behalf of Jiang Xu 
Sent: Tuesday, June 22, 2021 6:47:36 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] missing covalent bond between asparagine and
N-acetylglucosamine using coot carbohydrate module and phenix.refine
 

   External Sender:  Use caution.
   Hello guys,     I have a problem building and refining an xtal
structure. My protein has a N-glycosylation site and I want to add
N-acetylglucosamine and manose to my protein structure. I used Coot's
carbohydrate module and let Coot automatically build the sugar chain
to the electron density. It worked pretty well, but I noticed that
the bond between the amine group of asparagine and the C1 of
N-acetylglucosamine is depicted as a dashed line, rather than a solid
line in Coot. After refinement of the structure using Phenix.refine, I
found the 

Re: [ccp4bb] missing covalent bond between asparagine and N-

[Rezaul Karim]

Hi Jiang,I can understand why you are confused. I think I faced such case once. 
But Paul & Folmer's description is correct, that's the way coot works. Here is 
the way I solved it.In coot, after linking save the pdb without 
hydrogen.(Optional, if Phenix says it needs cif file - do readyset)Refine that 
pdb in Phenix without hydrogen.Then check it in coot & pymol. Even coot shows 
dotted lines, pymol will show a solid bond!Hope it helps!

Best,Reza

Md Rezaul Karim, Ph.D.
Postdoctoral Research Fellow
Department of Drug Discovery
H. Lee Moffitt Cancer Center and Research Institute
Tampa, FL 33612
Email: reza.ka...@moffitt.org, rez...@usf.edu
Phone: (813) 745 4673 ext. 5462
https://orcid.org/-0002-0424-127X 
 
  On Thu, Jun 24, 2021 at 7:40 AM, jiang wrote:   Hello 
guys,     I am a little bit confused. Since the polysaccharide is attached to 
asparagine via covalent bond. I am not sure what “link” means anyway.  Does it 
mean some other chemical bonds? Or not chemical bond at all.      As you can 
see from the book link below, the nitrogen atom  from the N-glycosylation site 
has covalent bond with only one hydrogen atom. The bond is referred to as 
glycosidic bond. https://www.ncbi.nlm.nih.gov/books/NBK22521/     
Thank you,Best,Jiang
Sent from my iPhone

On Jun 24, 2021, at 12:35 AM, Folmer Fredslund  wrote:



Dear Jiang
As Paul Emsley was already kind to point out:
Coot draws solid lines for bonds between neighbouring monomers in a polymer.
The bond between NAG and ASN is not such a bond - it is a link.
Links are drawn as dashed lines in Coot.

So, a dashed line is simply how such a bond is represented in Coot. It's not a 
bug in the software.
I'm not quite sure why you want the bond to be represented with a solid line 
instead of a dashed line. It makes no difference whatsoever on the validity of 
the model.
Best wishesFolmer Fredslund





ons. 23. jun. 2021 01.23 skrev Jiang Xu :

Hello David,    I used the "Make Link" under the "Modeling..." tab to create 
the bond. It worked, but the bond displayed is still a dashed line, not a solid 
line as shown below. I still don't know how to use phenix to add a solid bond 
between the two atoms.  So, is it a bug in the software or I didn't do it 
correctly? 
Thank you,Best,Jiang
On Tue, Jun 22, 2021 at 3:48 PM Jiang Xu  wrote:

Hello David,    I tried to rebuild the polysaccharide chain using the N-link 
add NAG, NAG, BMA method, saved the model and ran phenix.refine without 
checking the "automatically add hydrogens to the model". I also double checked 
to make sure that the "Link carbohydrates to protein and other carbohydrates" 
within the "Automatic Linking Options" was checked. The "Carbohydrate bond 
cutoff" is the set to the default value of 1.99, which I didn't change.  
However, after running phenix.refine, the bond disappeared again. As shown 
below, on the left is the input model, on the right is the refined output. You 
can see that the bond is gone and the distance between C1 of NAG and the 
Nitrogen atom of the amine group is 1.42 A. So Could anything else cause the 
problem?
Thank you,Best,Jiang
On Mon, Jun 21, 2021 at 11:11 PM David Briggs  wrote:

Hi Jiang Xu,

(Phenixbb added as I suspect this is Phenix not coot that has caused your issue)

Did you check the "add hydrogens" button in Phenix.refine? Sometimes this can 
erroneously add a second H to the ND of glycosylated Asn residues. (I assume 
this is because bond distances in the input PDB file fall outside Phenix's 
definitions of an Asn-NAG link).
If the second H is added, this breaks the refinement of the Asn-NAG bond.

My work around is to run "Ready Set" to add hydrogens as a separate job, and 
then manually inspect and correct the Asn residues as necessary before running 
Phenix.refine and _not_ checking the "add hydrogens" box.
I hope this fixes your problem.
Good luck,
Dave
--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs
From: CCP4 bulletin board  on behalf of Jiang Xu 

Sent: Tuesday, June 22, 2021 6:47:36 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] missing covalent bond between asparagine and 
N-acetylglucosamine using coot carbohydrate module and phenix.refine  External 
Sender: Use caution. Hello guys,    I have a problem building and refining an 
xtal structure. My protein has a N-glycosylation site and I want to add 
N-acetylglucosamine and manose to my protein structure. I used Coot's 
carbohydrate module and let Coot automatically build the sugar chain to the 
electron density. It worked pretty well, but I noticed that the bond between 
the amine group of asparagine and the C1 of N-acetylglucosamine is depicted as 
a dashed line, rather than a solid line in Coot. After refinement of the 
structure using Phenix.refine, I found the bond just disappeared, and the amine 
group still has two hydrogen atoms, which should contain 

[ccp4bb] Last days to register: PSB Symposium "Frontiers in Bioimaging", 1-2 July 2021 - FREE registration, deadline 27 June

[Daouda Traore]

Hi all,

Last days to register (*It's FREE.* *Deadline on 27 June 2021*) 
https://www.esrf.fr/psbsymposium2021 


The symposium includes an exciting collection of invited speakers, 
selected talks, interactive virtual posters, and interactive virtual 
lounges.


Looking forward to seeing you at the Symposium,

The organising committee

On 2021-06-14 16:12, PSB Symposium 2021 wrote:


Dear all,

The Partnership for Structural Biology (PSB), an alliance of research 
institutes (EMBL, ESRF, IBS and ILL) located in Grenoble, France, is 
pleased to announce that the PSB Symposium "Frontiers in Bioimaging" 
will take place on 1-2 July 2021. The meeting will be held remotely on 
a virtual event platform over two afternoons, and will include a 
series of invited and selected talks, interactive virtual posters, and 
interactive virtual lounges.


This is to announce that registration is still opened (*It's FREE.* 
*Deadline on 27 June 2021*) and the programme is available.


For further information and to check out the exciting collection of 
invited speakers: https://www.esrf.fr/psbsymposium2021 



The aim and scope of this meeting is to highlight progress in 3D 
imaging research that bridges the gap between the atomic and cellular 
scales, with spatial resolutions spanning from subnanometer to 
submicrometer range. Featured topics will include: cryo-electron 
tomography, X-ray tomography, volume electron microscopy, image 
analysis, super-resolution microscopy, and correlative approaches. The 
applications of the above methods to address essential questions in 
life sciences is of particular interest.


Best regards,

The organising committee





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Re: [ccp4bb] missing covalent bond between asparagine and N-acetylglucosamine using coot carbohydrate module and phenix.refine

[jiang]

Hello guys,
 I am a little bit confused. Since the polysaccharide is attached to 
asparagine via covalent bond. I am not sure what “link” means anyway.  Does it 
mean some other chemical bonds? Or not chemical bond at all.
  As you can see from the book link below, the nitrogen atom  from the 
N-glycosylation site has covalent bond with only one hydrogen atom. The bond is 
referred to as glycosidic bond.
 https://www.ncbi.nlm.nih.gov/books/NBK22521/
 
Thank you,
Best,
Jiang
Sent from my iPhone

> On Jun 24, 2021, at 12:35 AM, Folmer Fredslund  wrote:
> 
> Dear Jiang
> 
> As Paul Emsley was already kind to point out:
> 
> Coot draws solid lines for bonds between neighbouring monomers in a polymer.
> The bond between NAG and ASN is not such a bond - it is a link.
> Links are drawn as dashed lines in Coot.
> 
> 
> So, a dashed line is simply how such a bond is represented in Coot. It's not 
> a bug in the software.
> 
> I'm not quite sure why you want the bond to be represented with a solid line 
> instead of a dashed line. 
> It makes no difference whatsoever on the validity of the model.
> 
> Best wishes
> Folmer Fredslund
> 
> 
> 
> 
> 
> 
> ons. 23. jun. 2021 01.23 skrev Jiang Xu :
>> Hello David,
>> I used the "Make Link" under the "Modeling..." tab to create the bond. 
>> It worked, but the bond displayed is still a dashed line, not a solid line 
>> as shown below. I still don't know how to use phenix to add a solid bond 
>> between the two atoms.  So, is it a bug in the software or I didn't do it 
>> correctly? 
>> 
>> 
>> Thank you,
>> Best,
>> Jiang
>> 
>>> On Tue, Jun 22, 2021 at 3:48 PM Jiang Xu  wrote:
>>> Hello David,
>>> I tried to rebuild the polysaccharide chain using the N-link add NAG, 
>>> NAG, BMA method, saved the model and ran phenix.refine without checking the 
>>> "automatically add hydrogens to the model". I also double checked to make 
>>> sure that the "Link carbohydrates to protein and other carbohydrates" 
>>> within the "Automatic Linking Options" was checked. The "Carbohydrate bond 
>>> cutoff" is the set to the default value of 1.99, which I didn't change.  
>>> However, after running phenix.refine, the bond disappeared again. As shown 
>>> below, on the left is the input model, on the right is the refined output. 
>>> You can see that the bond is gone and the distance between C1 of NAG and 
>>> the Nitrogen atom of the amine group is 1.42 A. So Could anything else 
>>> cause the problem?
>>> 
>>> 
>>> 
>>> Thank you,
>>> Best,
>>> Jiang
>>> 
 On Mon, Jun 21, 2021 at 11:11 PM David Briggs  
 wrote:
 Hi Jiang Xu,
 
 (Phenixbb added as I suspect this is Phenix not coot that has caused your 
 issue)
 
 Did you check the "add hydrogens" button in Phenix.refine? Sometimes this 
 can erroneously add a second H to the ND of glycosylated Asn residues. (I 
 assume this is because bond distances in the input PDB file fall outside 
 Phenix's definitions of an Asn-NAG link).
 
 If the second H is added, this breaks the refinement of the Asn-NAG bond.
 
 My work around is to run "Ready Set" to add hydrogens as a separate job, 
 and then manually inspect and correct the Asn residues as necessary before 
 running Phenix.refine and _not_ checking the "add hydrogens" box.
 
 I hope this fixes your problem.
 
 Good luck,
 
 Dave
 
 --
 Dr David C. Briggs
 Senior Laboratory Research Scientist
 Signalling and Structural Biology Lab
 The Francis Crick Institute
 London, UK
 ==
 about.me/david_briggs
 
 From: CCP4 bulletin board  on behalf of Jiang Xu 
 
 Sent: Tuesday, June 22, 2021 6:47:36 AM
 To: CCP4BB@JISCMAIL.AC.UK 
 Subject: [ccp4bb] missing covalent bond between asparagine and 
 N-acetylglucosamine using coot carbohydrate module and phenix.refine
  
  
 External Sender: Use caution.
  
 Hello guys, 
I have a problem building and refining an xtal structure. My protein 
 has a N-glycosylation site and I want to add N-acetylglucosamine and 
 manose to my protein structure. I used Coot's carbohydrate module and let 
 Coot automatically build the sugar chain to the electron density. It 
 worked pretty well, but I noticed that the bond between the amine group of 
 asparagine and the C1 of N-acetylglucosamine is depicted as a dashed line, 
 rather than a solid line in Coot. After refinement of the structure using 
 Phenix.refine, I found the bond just disappeared, and the amine group 
 still has two hydrogen atoms, which should contain one hydrogen atom.  So, 
  how to solve this problem? 
 
 
 Thank you,
 Best,
 Jiang Xu
 Lin Chen's Research Group
 University of Southern California
 
 To unsubscribe from the CCP4BB list, click the following link:
 https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
 
 

[ccp4bb] E.coli RNA polymerase

[vipul panchal]

Dear All,

First of all, excuse me for writing an off-topic subject.

Our group is working on high throughput screening of a non-coding RNA to
investigate its therapeutic potential. We require *E.coli* RNA polymerase
and *E.coli* sigma-70 to perform In-vitro transcription for bulk synthesis
of ncRNA.
NEB has offered us a price of 84,000 EUR for 50ml of E.coli RNA pol
saturated with sigma-70. This is still expensive for us.

I am writing here hoping for a potential collaboration with a group who
routinely purifies *E.coli* RNA pol and sigma-70. If this is possible,
kindly let me know.

Best wishes,
Vipul
-- 
Vipul Panchal, PhD
University of Bergen
(M)-+47 46359545



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