Denaturing Gradient Gel Electrophoresis (DGGE)

Biology

Your institution must subscribe to JoVE's Biology section to access this content.

Fill out the form below to receive a free trial or learn more about access:

 

Cite this Article

Copy Citation | Download Citations

Peterson, C. Denaturing Gradient Gel Electrophoresis (DGGE). J. Vis. Exp. (2), e164, doi:10.3791/164 (2007).

Abstract

Disclosures

The authors have nothing to disclose.

Comments

9 Comments

  1. Hi Celeste, good day. thank you for the informative demonstration and discussion. May I know if DGGE is  applicable  in analyzing  plant diversity without prior sequence  information?   Many thanks in advance. yoky  

    Reply
    Posted by: Anonymous
    July 2, 2008 - 6:25 AM
  2. This movie was made with very un-professional behavior. You need to read Biorad DGGE protocol first and watch their movie clip. Also you need to fill the solution into the tubing (before triangle mixer). The length of tubing is longer than usual size.  

    Reply
    Posted by: Anonymous
    August 19, 2008 - 4:11 PM
  3. where is the spacer alignment plate? Very funny 

    Reply
    Posted by: Anonymous
    August 19, 2008 - 4:14 PM
  4. Thank you, celesteA²81;  

    Reply
    Posted by: Anonymous
    August 21, 2008 - 6:11 AM
  5. Hi,   Recently we bought the DCode System from Bio-Rad for DGGE analysis. The research goal is to analyze the stool microflora composition by DGGE and I have been trying to get our PCR condition with 16s rRNA variable region-specific primer sets optimized. But the results were not satisfactory because many nonspecific bands were amplified from the PCR reactions. I just started this project so I do not have much experience in this field. Therefore I will be very happy if you could give me some tips to solve the problem. DGGE Trouble Shoot!   For nested PCR first we used the bacterial 16S r RNA universal primer to amplify the genomic DNA from the stool samples. Those PCR products were used as a template for the V3 region amplification to observe the DGGE band pattern. Prior to run in DGGE gel, we tried to run in the agarose gel to see the primer specificity, but we could not get a specific single band.     First PCR Primer Set       Second PCR / DGGE Primer Set: C            
    Non-specific band   Expected band with non-specific background       Lane 1: Direct PCR with DGGE Primer set Lane ²: Nested PCR with DGGE Primer set All of the PCR mixture was prepared in different conditions of following range of PCR components: PCR Mixture Components: DNA Template (10 to ²00 ng) Primer F (5-²5 pmol) Primer R (5-²5 pmol) PCR Buffer (To make final 1X) dNTP (final concentration 0.1-0.3mM) Sterile dH²O (To make final Vol ²0 ul) DNA Tag Polymerase (1unit to ² unit) MgCl² (To make final 1mM to 6mM)                         I found that nested PCR can often generate secondary, non-specific bands. We tried to optimize PCR conditions (different amounts of primer, temp 55OC to 64OC, cycle no ²8 to 35, magnesium concentration 1mM to 6mM) using GO Tag DNA polymerase from Promega and Platinum Tag DNA polymerase from Invitrogen to get a specific band. We couldn’t achieve our aims to get specific band of PCR product with or without nested PCR, we failed to get it!     Any information on getting specific bands using DGGE primer set would be highly appreciated.   Thanks in advance!   Sicnerely, Samudra Acharya, Ph.D. Research Associate, R& D Center of Cellbiotech, S.Korea samudra@cellbiotech.com      

    Reply
    Posted by: Anonymous
    February 16, 2009 - 5:47 AM
  6. This VIDEO not open very well interrupted many times

    Reply
    Posted by: Anonymous
    March 12, 2009 - 3:25 AM
  7. Vishal, please send us an email at support@jove.com and we try to figure out why you have poor performance.

    Reply
    Posted by: Anonymous
    April 16, 2009 - 6:44 PM
  8. I only want to know the brand of your system, because we would like to have the same comb for our gels

    Reply
    Posted by: klopp p.
    October 5, 2009 - 9:22 AM

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Usage Statistics