Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Animal and Molecular Model Systems > Yeast Forum
Register Search Today's Posts Mark Forums Read

Yeast Forum Discuss research on yeast the model organism. Post questions and topics on the molecular biology and genetics of Saccharomyces cerevisiae and other yeast species.


2 hybrid library amplification and plasmid isolation problems

2 hybrid library amplification and plasmid isolation problems - Yeast Forum

2 hybrid library amplification and plasmid isolation problems - Discuss research on yeast the model organism. Post questions and topics on the molecular biology and genetics of Saccharomyces cerevisiae and other yeast species.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 08-17-2005, 05:04 AM
Gregory Finnigan
Guest
 
Posts: n/a
Default 2 hybrid library amplification and plasmid isolation problems



I am a new graduate student rotating in a yeast lab and I am having
trouble amplifying the James genomic library (C1,2,&3) for a 2-hybrid
screening using GAL4 as the construct. I have very old samples of the
plasmid DNA as well as new libraries in E. coli. I have tried
amplifying the libraries (from the old plasmid DNA) using TOP10
electrocompetent E. coli and cannot get a high enough transformation
efficiency. Thus, with new libraries (in E. coli) I am trying to
fomulate a protocol for amplifying the 5 x 10^6 clones properly and
isolating the plasmids (at least 100-500 ug DNA). Thus far, using the
QIAGEN HiSpeed Midi-Prep Kit I have only been able to extract 5 ng/ul
plasmid. Is there any protocol for amplification as well as
hints/suggestions for getting higher yields on large-scale plasmid
preps? Thanks in advance for any help!

G

Reply With Quote
Reply

Tags
amplification , hybrid , isolation , library , plasmid , problems


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
how the amplification length is determined in terms of genomic or plasmid DNA in PCR? badly need PCR - Polymerase Chain Reaction Forum 1 01-12-2010 04:56 PM
Semi Solid Attempt 2 Holt, Ben F. III Arabidopsis and Plant Biology 0 04-04-2009 11:00 PM


All times are GMT. The time now is 10:19 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 0.10713 seconds with 16 queries