Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


E. coli electroporation efficiencies - pBR322 v/s pUC ori

E. coli electroporation efficiencies - pBR322 v/s pUC ori - Protocols and Methods Forum

E. coli electroporation efficiencies - pBR322 v/s pUC ori - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 06-25-2006, 02:13 PM
Haviland, David L
Guest
 
Posts: n/a
Default E. coli electroporation efficiencies - pBR322 v/s pUC ori



AC:

I've found differences as well in transformation effeciency, also based on size. But at the same time I've not rechecked the origin of replication of the respective vectors.

Currently, TOP10F's, and SURE cells have been our bugs of choice so that is where my current experience is.

By electroporation, we have easily obtained 10^9 cfu/ug of pUC19 with both cells. But as we know, this is also a small vector. After knowing this, I then tried a nubmer of vectors we had in the lab. pBluescript at 3kb rendered high 10^8 cfu/ug. pCDNA3 at 5.3kb was in the low 10^8 cfu/ug. With a 7 kb insert, for a total of 12.3 kb, we had plummeted to the low 10^7 cfu/ug. The only other thing I had was a cosmid, checking in around 47kb (7kb for pTCF vector and about 40kb for the insert) the effiency plummeted to the mid 10^4 cfu/ug.

I could be wrong but if memory serves most of these are using the old pBR322 backbone with its ORI of replication.

So by comparison, I think we have similar results with different sized vectors.

David



-----Original Message-----
From: [Only registered users see links. ] on behalf of Aawara Chowdhury
Sent: Sat 6/24/2006 4:02 PM
To: [Only registered users see links. ]
Subject: E. coli electroporation efficiencies - pBR322 v/s pUC ori

Is transformation by electroporation affected into E. coli affected
by the replication origin on the plasmid?

We test our electrocompetent DH5alpha using a 7.7 kb plasmid, and
get efficiencies around 3x10e7/ug - 4x10e7/ug.

Adjusting for plasmid size, this translates into about 1x10e8/ug for
pUC18 or pUC19, which several commercial suppliers of competent cells
use as a standard. I'm curious why we never achieve the > 5x10e8
transformants per ug that commercial suppliers claim in their literature.

Does this have anything to do with the replication origin on the plasmid?
The plasmid we use to test efficiency has the pMB1 origin (which has
the rop gene, and is present at 10 - 30 copies/cell). I know that pUC
is at a much higher copy number (>300 copies/cell).

If copy number does affect the ability of a transformed cell to form
an antibiotic-resistant colony, then I can believe that our competent
bacteria are about as good as can be made.

AC

P.S. Note - we have made other strains of E. coli competent to
significantly different degrees (easily exceeding 1x10e9/ug for MC1061,
or being as low as 1x10e7/ug for STBL2).
--
Email: echo 14232209320335931527179462016806497532155427589018 6P | dc
_______________________________________________
Methods mailing list
[Only registered users see links. ]
[Only registered users see links. ]


Reply With Quote
  #2  
Old 06-25-2006, 08:57 PM
Aawara Chowdhury
Guest
 
Posts: n/a
Default E. coli electroporation efficiencies - pBR322 v/s pUC ori

Hi David,

Thanks for your response. Actually, all the plasmids you've used have
high copy origins (in the sense that none of them have the "rop" gene).
All of them (including pUC) are essentially rop-deleted derivatives of
the pMB1 origin that is in pBR322.

Your experience does indicate to me that the relationship between size
and electroporation efficiency is not linear (even in the 2.5 kb - 12.5 kb
range).

Thanks,
AC

In <[Only registered users see links. ].net >,
Haviland, David L <[Only registered users see links. ].edu> wrote:



--
Email: echo 14232209320335931527179462016806497532155427589018 6P | dc
Reply With Quote
  #3  
Old 06-26-2006, 03:08 AM
DK
Guest
 
Posts: n/a
Default E. coli electroporation efficiencies - pBR322 v/s pUC ori

In article <2NCng.6818$[Only registered users see links. ]>, [Only registered users see links. ] wrote:

Yes, it is not linear at all. It goes down rather steeply (exp?) with the
size. Also, the function "efficiency/ug" vs the actual DNA amount
used is highly non-linear, so when one tests using 1 ng of plasmid
usually (always in my hands) does not give the same number
as 1 pg.

DK
Reply With Quote
  #4  
Old 06-26-2006, 03:18 AM
DK
Guest
 
Posts: n/a
Default E. coli electroporation efficiencies - pBR322 v/s pUC ori

In article <[Only registered users see links. ].net >, "Haviland,
David L" <[Only registered users see links. ].edu> wrote:


Just FYI: for huge DNAs, a new factor comes into consideration.
15-20 kV/cm intensity is high enough to cause DNA melting. As a
result, large DNAs have good chance to tangle up (much like
chromosomal DNA in alkiline lysis). This effect has been demonstrated
experimentally (I could find a ref. if needed). With this in mind, it is not
surprising that empirically it was found that when electroporating
very large DNAs, one is better off using 10-12.5 kV/cm than 15-20 kV/cm
and pulses of 3-5 ms that give highest efficiency for small plasmids
(similarly, I provided this ref here long time ago but don't have
it now).

DK
Reply With Quote
Reply

Tags
coli , efficiencies , electroporation , ori , pbr322 , puc , v or s


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Electroporation optimization guide DK Protocols and Methods Forum 0 08-09-2007 11:39 PM
Question about pBR322 and pUC Tim Fitzwater Protocols and Methods Forum 0 07-10-2007 02:43 PM
Question about pBR322 and pUC Liansen Liu Protocols and Methods Forum 0 07-09-2007 04:51 PM
E. coli electroporation efficiencies - pBR322 v/s pUC ori Aawara Chowdhury Protocols and Methods Forum 0 06-24-2006 09:02 PM


All times are GMT. The time now is 02:48 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.14048 seconds with 16 queries