Methods Digest, Vol 63, Issue 7-Plasmid selection/maintainance sans-Antibiotic

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Sat Aug 21 07:27:58 EST 2010


why one should make the procedures difficult to handle using X-amino
acid deficient E.coli to complement with plasmid containing X-amino
acid synthase as it will require use of synthetic auxotrophic media
instead of simple LB. If you have specific requirement it can be tried
subject to media meeting complete nutritional requirement of E.coli
less X amno acid.

On Fri, Aug 20, 2010 at 10:36 PM,  <methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. Plasmid selection/maintainance sans-Antibiotic (Cathal Garvey)
>   2. induction and overexpression of a 7 kDa proteins in E coli
>      (venkatramanan rao)
>   3. RE: phospho-Akt blotting (Bari Zahedi)
>   4. Re: induction and overexpression of a 7 kDa proteins in E
>      coli (David-Paul Minde)
>   5. Re: induction and overexpression of a 7 kDa proteins in E
>      coli (Zhonglin Chai)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Thu, 19 Aug 2010 16:53:03 +0100
> From: Cathal Garvey <cathalgarvey from gmail.com>
> Subject: Plasmid selection/maintainance sans-Antibiotic
> To: "Methods @ Bio.net" <methods from magpie.bio.indiana.edu>,
>        methods from oat.bio.indiana.edu
> Message-ID:
>        <AANLkTim2xnfBGQ3NWnddcXcQgHvAPvQmmOEZS8GyvkCL from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hello all,
> I am considering the development of a plasmid-based bacterial
> transformation/expression vector that would sidestep the use of antibiotic
> selection in a range of cell types. I was hoping someone on this list could
> offer some insight into current methods that avoid the use of antibiotics?
>
> I know some methods call for the use of strains lacking the ability to grow
> on media without X amino acid, and complement this deficiency with a
> plasmid-encoded X-synthase. While this is very clever, it does restrict work
> to appropriate strains only, which is too inflexible for me.
>
> What else is out there, besides simply diluting and streaking cells hoping
> for a positive single colony?
> Thanks,
> Cathal Garvey
>
> --
> letters.cunningprojects.com
> twitter.com/onetruecathal
> twitter.com/labsfromfabs
>
>
> ------------------------------
>
> Message: 2
> Date: Thu, 19 Aug 2010 11:01:34 -0700
> From: venkatramanan rao <ventigers from gmail.com>
> Subject: induction and overexpression of a 7 kDa proteins in E coli
> To: methods from magpie.bio.indiana.edu
> Message-ID:
>        <AANLkTimn-xbBHDjD0UNKA8PPcuYSAmgfHnXaBb4oOCaH from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hello all,
> I want to do domain mapping of my protein of intrest for testing its
> interaction with a  few predicted interactors. I have generated
> N-termius end and C-terminus end of my protein. I have cloned
> approx150 bp both the terminii in pET28a vector between Nde1 and EcoRI
> sites(both the constructs). I tried inducing the protein in BL-21DE3
> cells using but no success. The full length protein expresses normally
> without any problem in a different vector backbone(infact gives a big
> blob of pure protein in induced cells) . I am wondering what went
> wrong. I have sent my  plasmid samples for sequencing and  awaiting
> for results. The constructs were PCR amplified using Pfu
> polymerase(very less chances of errors in PCR product). Since I have
> done directional cloning chances of frameshift is also less. I tried
> for presence of inserts using both colony PCR and plasmid as template
> both methods show presence of template of correct size. I was
> wondering if anybody has expressed protein of similar size. Is it
> getting degraded. Please help me in this regard.
>
> --
> Rao Venkatramanan G.
> Research Scholar
> UM-DAE-CBS
> Mumbai University campus,
> Kalina, Santacruz
> Mumbai-400098
>
>
>
> ------------------------------
>
> Message: 3
> Date: Thu, 19 Aug 2010 11:28:06 -0700
> From: Bari Zahedi <bzahedi from bccrc.ca>
> Subject: RE: phospho-Akt blotting
> To: Chris McDermott-Roe <cjmcdermottroe from googlemail.com>,
>        "methods from magpie.bio.indiana.edu" <methods from magpie.bio.indiana.edu>
> Message-ID:
>        <B2B905CD6A0E134E9C2347E3B7BF7C8C02F06FED5F25 from crcmail4.BCCRC.CA>
> Content-Type: text/plain; charset="us-ascii"
>
> Hi Chris, i've never looked at cardiamyocytes. But i do westerns for pAKT all the time and i feel your pain! But i have gotten them to work routinely.
>
> i don't have a lot of time today so i'll answer you quickly and email me if you need more info.
>
> -you need at least over 10  and most likely over 20ug of protein loaded to see a good signal. It is great if you are stimulating your cells because then you can tell if your problem is low abundance of pAKTgenerated by your system vs. due to your western conditions. So if you stimulate (activate) a cell surface receptor upstream of AKT and compare nil to stimulated, you should see an increase in pAKT.
>
> - you can always use Jurktat Ts as a control as well, their pAKT levels are high.
>
> - i am currently using the pAKT ab from Invitrogen because the last few batches of the Cell Signalling ab have been crap. if you are using this ab and have bought it in the past few months, try to get your money back. a bunch of us did. If you are using the older version of the Cell signalling ab ( ie they now have a new set of Abs they sell). try using it as a 1/500 overnight in 2% BSA.
> -If you are using non fat milk, be carefull, the stuff from the store has phosphatases in it and wipes out the pakt signal. even though they don't recommend it, most of us at my work that are successfull with pakt staining where it is weak use bsa. you can always order phosphatase free nonfat milk. someone sells it, not sure who.
> - Also the Cell signalling should send you a aliquot of their new ab. it works nicely, but you can only use it once ( we usually reuse our Abs and keep them in azide at 4 degrees for months at a time-you have to test this for each specific Ab though). Do not try to use it more than once if your pAKT levels are low in your system because you will be cursing and disappointed!
> - what i do for my western protocol:
> block membrane n 5% bsa for 20-30 min no more
> add 1/1000 Invitrogen pAKT ( i can't remember the code but it says its for 10 reactions on the website incase you are looking it up) in 2%BSA+ Azide+TBST put at 4degreesC overnight- RT doesn't work well.
> Wash next day with TBST 10minX3.
> Add secondary as you usually would at RT for 1 hour.
> wash same as before and detect.
> I sometimes get a non specific band which is MUCH more visible than my pAKT which will be the weak stuff below this band. This could be only in my cells though as some of my friends who work with other cell types don't get this.
> Good luck!
>
>
>
>
>
> ________________________________________
> From: methods-bounces from oat.bio.indiana.edu [methods-bounces from oat.bio.indiana.edu] On Behalf Of Chris McDermott-Roe [cjmcdermottroe from googlemail.com]
> Sent: August 19, 2010 5:38 AM
> To: methods from magpie.bio.indiana.edu
> Subject: phospho-Akt blotting
>
> Hi everyone,
>
> Can somebody provide me with a robust blotting protocol for phospho-Akt (in
> cardiacmyocytes preferably)?  If you could tell me which Ab and blotting
> conditions work best, that would be really excellent!!  Safe to say I'm
> tearing my hair out in frustration.
>
> thanks as always
>
> Chris
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
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>
>
>
> ------------------------------
>
> Message: 4
> Date: Thu, 19 Aug 2010 21:24:22 +0200
> From: David-Paul Minde <davidminde from gmail.com>
> Subject: Re: induction and overexpression of a 7 kDa proteins in E
>        coli
> To: venkatramanan rao <ventigers from gmail.com>
> Cc: methods from magpie.bio.indiana.edu
> Message-ID:
>        <AANLkTimRXCmf1wcsVJ_xmj8dACpsZoaOqwyyAL=MoE+9 from mail.gmail.com>
> Content-Type: text/plain; charset=UTF-8
>
> ... for peptides/miniproteins SMT3 (i.e. yeast "SUMO") fusion can be
> extremely useful. Usually, it helps avoiding degradation. Yields up to
> some 100 mg pure peptide per liter  :=)
> have fun,
> David Minde (Drs)
>
> Cellular Protein Chemistry Room 707
> Department of Chemistry
>  Faculty of Science
>  Utrecht University
> Krytgebouw
>  Padualaan 8
>  NL-3584 CH Utrecht
>  The Netherlands
>
> office phone +31 30 253 4105
> (magic:) android  +31 652614443
> ... "Mannschaftsgeist" :)
> Science is what happens while we are making other plans (~John Lennon)
>
>
>
> ------------------------------
>
> Message: 5
> Date: Fri, 20 Aug 2010 08:34:00 +1000
> From: Zhonglin Chai <Zhonglin.Chai from bakeridi.edu.au>
> Subject: Re: induction and overexpression of a 7 kDa proteins in E
>        coli
> To: venkatramanan rao <ventigers from gmail.com>
> Cc: "methods from magpie.bio.indiana.edu" <methods from magpie.bio.indiana.edu>
> Message-ID: <231F3618-6D4C-46D5-9A19-9E8284247FFE from bakeridi.edu.au>
> Content-Type: text/plain; charset="us-ascii"
>
> Possibility for this small protein to run out of your gel? PAGE
> condition? Do you have other method to detect the presence of your
> protein? ELISA?
>
> Sent from my iPhone
>
> On 20/08/2010, at 5:11 AM, "venkatramanan rao" <ventigers from gmail.com>
> wrote:
>
>> Hello all,
>> I want to do domain mapping of my protein of intrest for testing its
>> interaction with a  few predicted interactors. I have generated
>> N-termius end and C-terminus end of my protein. I have cloned
>> approx150 bp both the terminii in pET28a vector between Nde1 and EcoRI
>> sites(both the constructs). I tried inducing the protein in BL-21DE3
>> cells using but no success. The full length protein expresses normally
>> without any problem in a different vector backbone(infact gives a big
>> blob of pure protein in induced cells) . I am wondering what went
>> wrong. I have sent my  plasmid samples for sequencing and  awaiting
>> for results. The constructs were PCR amplified using Pfu
>> polymerase(very less chances of errors in PCR product). Since I have
>> done directional cloning chances of frameshift is also less. I tried
>> for presence of inserts using both colony PCR and plasmid as template
>> both methods show presence of template of correct size. I was
>> wondering if anybody has expressed protein of similar size. Is it
>> getting degraded. Please help me in this regard.
>>
>> --
>> Rao Venkatramanan G.
>> Research Scholar
>> UM-DAE-CBS
>> Mumbai University campus,
>> Kalina, Santacruz
>> Mumbai-400098
>>
>> _______________________________________________
>> Methods mailing list
>> Methods from net.bio.net
>> http://www.bio.net/biomail/listinfo/methods
>>
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> End of Methods Digest, Vol 63, Issue 7
> **************************************
>



-- 
Dr V K Gupta
Sr Microbiologist (Mol Biology)
IMBL, Department of Entomology
Pun. Agric. Univ., Ludhiana (Pb)-141004- India
M: 081465-55515



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