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

DNA Techniques Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


DNA Ligations Factors Affecting Efficiency

DNA Ligations Factors Affecting Efficiency - DNA Techniques

DNA Ligations Factors Affecting Efficiency - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 09-17-2007, 07:20 AM
kiki06's Avatar
Post-Doc
Points: 5,313, Level: 49 Points: 5,313, Level: 49 Points: 5,313, Level: 49
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2006
Posts: 277
Thanks: 0
Thanked 6 Times in 5 Posts
Cool DNA Ligations Factors Affecting Efficiency



Hello guys,

Factors affecting DNA Ligation efficiency

Optimal Ligation Effiency was using:
  • 50 ng of vector
  • Overnight Ligation
  • 16°C
  • 2:1 insert:vector molar ratio
  • standard T4 ligase used
  • Ligase was heat inactivated at 65°C for 20 mins
  • 2 μL / 20 μL was used to transform commercial heat-shock competent cells.
(7.2 kb vector + 0.6 kb insert with both sticky ends).



Now What affects Ligation Efficiency:


DNA ligation efficiency was marginally decreased by


  • Using 100 ng vector (more DNA is not better!)
  • Doing a 1 hr ligation at room temperature
  • Using insert:vector molar ratios of 5:1 and 1:1



Ligation efficiency was noticably decreased (x100) by
  • Sticky end ligation with a larger insert (5.2 kb vector + 2.6 kb insert)
  • Blunt end ligation
Ligation efficiency was severely decreased (x10000) by
  • Using DNA fragments that have been exposed to ethidium bromide and UV during the gel extraction procedure (difficult to avoid but heartily recommended)
  • Using the NEB Quick Ligation Kit (possibly a bad batch)
If anyone can post more ideas here that would be great

Kiki
Reply With Quote
Reply

Tags
affecting , dna , efficiency , factors , ligations


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
Help me. What is tumorgenesie Subpressing Factors? minhnv0312 Biology Forum 0 04-13-2010 05:03 AM
qPCR NEWS December 2008 Editor www.Gene-Quantification.info Protocols and Methods Forum 0 12-17-2008 09:03 AM
Efficiency of (electro)transformation in Salmonella C Coward Protocols and Methods Forum 1 11-28-2008 05:11 PM
q PCR efficiency Ivan Real-Time PCR and Quantitative PCR Forum 3 09-05-2008 01:38 PM
qPCR newsletter August 2008 with focus on PCR efficiencydetermination Editor www.Gene-Quantification.info Protocols and Methods Forum 0 08-14-2008 01:38 PM


All times are GMT. The time now is 03:20 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 0.11961 seconds with 16 queries