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 55^OC to 64^OC, 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