Dear Frauke,

I don't think you have to worry too much. You want to attach a
C-terminal GFP tag, which is easy to monitor in vivo. If there is a
frame shift problem upon recombination in some of the clones, you will
not see GFP, therefore just pick those clones that express the GFP
fusion and verify later on a Western blot.

Regards,


Dr. Francisco Rivero
Centre for Cardiovascular and Metabolic Research
The Hull York Medical School
University of Hull
HU6 7RX Hull
UK
Phone: +44 1482 466433
e-mail: [log in to unmask]



-----Original Message-----
From: DICTY [mailto:[log in to unmask]] On Behalf Of
Frauke Bach
Sent: 18 August 2011 15:54
To: [log in to unmask]
Subject: [DICTY - 621] Generation of knock-in construct

Dear Dicty community,

We are planing to knock in a gfp tag downstream a gene of interest.
Therefore the upstream flank of the construct lies in the coding region
of the gene. To generate the knock-in construct we had to introduce a
restriction site at the 5'end of the upstream flank.
Our question is, if the few additional basepairs of the restriction site
will also be integrated into the genome (even though, they are not part
of the homologous region). In our case this would lead to a frameshift
within our target gene which would be detrimental.
Are there easy ways to get around this problem (if no endogenous
restriction site is present)?
  
Greetings from Hamburg,
Frauke


Bernhard-Nocht-Institute for Tropical Medicine
Research Group Hagedorn
[log in to unmask]


*****************************************************************************************
To view the terms under which this email is distributed, please go to http://www.hull.ac.uk/legal/email_disclaimer.html
*****************************************************************************************