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Denaturing Gradient Gel Electrophoresis (DGGE)
Celeste Peterson
Department of Microbiology and Molecular Genetics, Harvard Medical School

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0:01 Examples of applications1

0:21 Subscription Lock21

0:23 Preparation23

2:45 Assemble gel delivery165

5:54 Deliver gel354

6:52 Assemble gel apparatus412

8:29 Load samples and run the gel509

8:51 Discussion531

 

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Peterson C (2007). Denaturing Gradient Gel Electrophoresis (DGGE). JoVE. 2. http://www.jove.com/index/details.stp?id=164, doi: 10.3791/164
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05/14/2007 11:24:42 AM
Anonymous responded with a statement of type: Neutral
An excellent demonstration of how to set up DGGE, but why isn't she wearing a lab coat?!! As our departmental biological safety officer I am amazed that:
a) The work was undertaken without adequate protective clothing.
b) The unsafe work was filmed.
c) The video is available for download as a demonstration of what to do.

Seriously; you have opened yourself up to legal action should someone try to replicate this and become exposed to polyacylamide.
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05/14/2007 9:49:09 PM
nikita@simx.com responded with a statement of type: Neutral
Test
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07/02/2008 6:25:04 AM
yoky responded with a statement of type: Question

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

 

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08/19/2008 4:11:29 PM
Anonymous responded with a statement of type: Neutral

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.

 

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08/19/2008 4:14:33 PM
Anonymous responded with a statement of type: Neutral

where is the spacer alignment plate? Very funny 

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08/21/2008 6:11:20 AM
douhenan responded with a statement of type: Agree

Thank you, celeste!

 

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02/16/2009 5:47:39 AM
Samudra responded with a statement of type: Neutral

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

 

 

 

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03/12/2009 3:25:44 AM
vishal patil responded with a statement of type: Question

This VIDEO not open very well interrupted many times

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04/16/2009 6:44:42 PM
nikitab responded with a statement of type: Neutral

Vishal, please send us an email at support@jove.com and we try to figure out why you have poor performance.

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03/26/2009 8:01:17 AM
vishal responded with a statement of type: Neutral

please i want   to download this video please tell me how i !  

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10/05/2009 9:22:30 AM
paquik responded with a statement of type: Question
I only want to know the brand of your system, because we would like to have the same comb for our gels
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02/25/2007

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Journal of Visualized Experiments (JoVE) is an online research journal employing visualization to increase reproducibility and transparency in biological sciences.

 

ISSN 1940-087X

 

 

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