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.


What is the size of the plasmid DNA pCMV-GFP?

What is the size of the plasmid DNA pCMV-GFP? - DNA Techniques

What is the size of the plasmid DNA pCMV-GFP? - 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 03-19-2007, 02:22 PM
Pipette Filler
Points: 1,366, Level: 21 Points: 1,366, Level: 21 Points: 1,366, Level: 21
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2007
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default What is the size of the plasmid DNA pCMV-GFP?



What is the size of the plasmid DNA pCMV-GFP?
Reply With Quote
  #2  
Old 03-19-2007, 03:20 PM
Pipette Filler
Points: 1,347, Level: 21 Points: 1,347, Level: 21 Points: 1,347, Level: 21
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2007
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default

i dunno.....honestly
Reply With Quote
  #3  
Old 03-19-2007, 03:20 PM
Pipette Filler
Points: 1,347, Level: 21 Points: 1,347, Level: 21 Points: 1,347, Level: 21
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2007
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default

Plasmid containing pCMV-GFP was propagated in transformed Escherichia coli cells. E coli cells were grown in standard Luria Bertani medium at pH 7.0 to a cell density of ~3 to 4 × 109 cells/mL. Cells were harvested by centrifugation and the plasmid DNA was extracted and purified using a Qiagen Plasmid Maxi Kit (Qiagen, Santa Clarita, CA) as per the manufacturer?s recommended protocols. In brief, the plasmid purification procedure involved alkaline hydrolysis of the cells and isolation of the plasmid by binding to an anion-exchange resin of proprietary composition. The concentration of the purified plasmid preparation was 0.87 µg/µL determined using a UV-visible spectrophotometer (SpectraMax Plus, Molecular Devices, Sunnyvale, CA) using OD260 (optical density at wavelength 260 nm). The OD260/OD280 ratio was 1.964, indicating that the plasmid preparation was sufficiently pure and could be used for transfection purposes. Standard agarose gel electrophoresis on a 0.8% wt/wt agarose gel was conducted to investigate the plasmid structural integrity and revealed 2 major bands. The high-mobility band was attributed to the most compact or supercoiled form of plasmid DNA. The other band with low mobility indicated the overall nonsupercoil content in the plasmid preparation.

Formation of DNA-Anionic Liposome Complexes

For a single transfection experiment, 225 ng of DNA was diluted into a liposome suspension equivalent to 40 µg lipid in a centrifuge tube. Anionic lipoplex formation was achieved by addition of calcium chloride solution. Ca2+ ions were introduced into the formulation using stock solution of a 2 M calcium chloride solution that had been previously filter sterilized through a 0.2-µm filter. The amount of plasmid DNA was identical in all experiments irrespective of the Ca2+ concentrations (~7 mM to 132 mM) introduced into each complexation tube. The final volume of the lipoplex suspension applied to the cells was 300 µL. The complexes were incubated for 25 minutes at room temperature.

[Only registered users see links. ]
Reply With Quote
  #4  
Old 06-02-2010, 02:19 PM
Pipette Filler
Points: 12, Level: 1 Points: 12, Level: 1 Points: 12, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2010
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: What is the size of the plasmid DNA pCMV-GFP?

Hi

Where did you get the plasmid containing pCMV-GFP from? That would be veeery helpful!

Thanks a lot,
Isabel
Reply With Quote
Reply

Tags
dna , pcmvgfp , plasmid , size


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
problem with pWATERGATE plasmid ismawanto Molecular Cloning Forum 5 05-31-2010 10:30 AM
Plasmid prep issues peter Protocols and Methods Forum 4 04-07-2007 10:53 PM
Can the size of a supercoiled plasmid DNA be shan1946 DNA Forum 2 03-30-2007 02:37 PM
GNU units and units.dat; Units of Measurement and Unit Conversion James Redford Physics Forum 0 07-31-2005 12:08 PM
yeast plasmid preps johnspar@earthlink.net Yeast Forum 0 02-20-2004 02:13 AM


All times are GMT. The time now is 06:08 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 0.15068 seconds with 16 queries