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

Recombinant Protein Forum Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


forgot to add apmicillin

forgot to add apmicillin - Recombinant Protein Forum

forgot to add apmicillin - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 01-17-2009, 06:45 PM
Pipette Filler
Points: 24, Level: 1 Points: 24, Level: 1 Points: 24, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default forgot to add apmicillin



I induced expression of a his-tagged protein in E.coli by adding ice cold LB with IPTG for 2 hours (and changing incubation temperature to 30 degrees from 37). Later I realised that I forgot to add ampicillin and chloramphenicol to the induction media. Would you go on and try to purify the protein from these cells anyway?

it's my first week at this new job and I guess I don't want to admit already that I made a stupid mistake....
Reply With Quote
  #2  
Old 01-17-2009, 08:41 PM
Pipette Filler
Points: 591, Level: 11 Points: 591, Level: 11 Points: 591, Level: 11
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2008
Posts: 10
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: forgot to add apmicillin

I would not continue since your protein yield may be WAY lower than expected since you haven't selected for your gene expressing bacteria. It is far more annoying to go through the entire purification than to just remedy a pretty simple situation.

I'm not sure what type of job it is but in my experience it is better to just put in the extra time (assuming you aren't getting paid overtime) and fix it. You aren't very deep into a mistake (should only need to reinocculate culture and induce) and chances are every scientist you work with has done something silly more than once. Better to establish yourself as someone who has intergrity in their work rather than leaving yourself to be exposed as someone who tries to get by.
Reply With Quote
  #3  
Old 01-17-2009, 10:55 PM
Pipette Filler
Points: 24, Level: 1 Points: 24, Level: 1 Points: 24, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: forgot to add apmicillin

you got a point there...

I was just thinking that since the media is not totally devoid of ampicillin (I did have ampicillin and chloramphenicol in the LB before adding induction medium) and as the cells were shifted to lower temperature maybe they wouldn't lose the plasmid. Well, it is better to be safe than sorry...
Reply With Quote
Reply

Tags
add , apmicillin , forgot


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
Kapazitaet der Krematorien in Auchwitz bartelsg77@yahoo.com Forum Physik 53 05-21-2009 08:33 PM


All times are GMT. The time now is 01:52 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12162 seconds with 16 queries