Re: [ccp4bb] OFFTOPIC question "Two plasmids in one host cell"

[Artem Evdokimov]

Hi

An easy fix is to PCR amplify your low copy number plasmid (modern
polymerases have no issues amplifying up to 20 kB if you have a largeish
vector) and then sequence the product. Alternatively you can amplify just a
portion of interest, of course (you said sequence the plasmid so I went
with your exact request). The spectionmycin trick has already been proposed
- that also works well :)

Now, you also mentioned that you would like to quantify the plasmid - for
that a simple qPCR (or even a carefully tuned regular PCR) will do the
trick, or if you want to go seriously old school you can quantify it by
dilution, transformation, and colony counting against a known standard
plasmid.

Happy new year

Artem

On Wed, Dec 30, 2020, 4:35 AM Anamika Singh  wrote:

> Hi All,
>
> I have two constructs having different ori, p15ori and M13 ori, different
> promoters araBAD promoter and LacI, and different antibiotic resistance
> chloramphenicol and Ampicillin respectively.
> I am managed to get the transformants and getting the expected result
> after blunt digestion with the EcoRV enzyme. Since both the plasmids have
> the site for EcoRV. But the p15 ori has a low copy number that's why I am
> seeing the very faint band as compared to other plasmid in the sample.
>
> So I would like to know is there any way that I can quantify the low copy
> number plasmid.
> Because I am not able to sequence it with the specific primers it could be
> due to its low concentration.
>
> Please advise.
>
> Thank you.
> --
> Dr. Anamika Singh
> Post-Doctoral Fellow
> Silberman Institute of Life Sciences
> Hebrew University of Jerusalem, Israel
> No: 054-294-8036
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>



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Re: [ccp4bb] OFFTOPIC question "Two plasmids in one host cell"

[Darren Hart]

The pACYC vectors (probably your p15ori plasmid) is said to have a copy 
number of ~10 copies per cell while your other plasmid is likely in the 
hundreds - see:


https://blog.addgene.org/plasmid-101-origin-of-replication

The protein expression level from the lower copy number plasmid is 
usually a bit lower, but not always.


For sequencing, we use primers that are specific to the pACYC vector so 
it does not matter if there is a second higher copy plasmid present in 
the miniprep.


To increase the amount of pACYC vector for sequencing, you can always 
amplify the copy number by adding spectinomycin to a growing culture - 
this inhibits protein synthesis, but the low copy plasmids continue to 
replicate to hundreds of copies per cell.


Darren



On 30/12/2020 10:35, Anamika Singh wrote:

Hi All,

I have two constructs having different ori, p15ori and M13 ori, 
different promoters araBAD promoter and LacI, and different antibiotic 
resistance chloramphenicol and Ampicillin respectively.
I am managed to get the transformants and getting the expected result 
after blunt digestion with the EcoRV enzyme. Since both the plasmids 
have the site for EcoRV. But the p15 ori has a low copy number that's 
why I am seeing the very faint band as compared to other plasmid in 
the sample.


So I would like to know is there any way that I can quantify the low 
copy number plasmid.
Because I am not able to sequence it with the specific primers it 
could be due to its low concentration.


Please advise.

Thank you.
--
Dr. Anamika Singh
Post-Doctoral Fellow
Silberman Institute of Life Sciences
Hebrew University of Jerusalem, Israel
No: 054-294-8036



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--

**

Dr. Darren J. Hart,

CNRS Research Director, Institut de Biologie Structurale (IBS)
Unité Mixte de Recherche UMR5075 (CEA-CNRS-UGA)


Director, Integrated Structural Biology Grenoble (ISBG)
Unité Mixte de Service UMS3518 (CNRS-UGA-CEA-EMBL)

**

Email: darren.h...@ibs.fr
Tel: +33 4 57 42 85 86

Physical address: IBS/ISBG, 71 avenue des Martyrs, 38000 Grenoble, France

Postal address: IBS/ISBG, 71 avenue des Martyrs, CS 20192, 38042 
Grenoble, Cedex 9, France


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