DON'T PANIC - falw.vu

A GUIDE TO DGGE (V2) Page 2
Gel electrophoresis and cleanup ........................................................................................... 19
WHAT TO DO IF YOUR EXCISED BAND ISN’T PURE.................................................... 20
A PROBLEMS CHECKLIST....................................................................................................... 20
TROUBLESHOOTING AND ADVANCED TOPICS................................................................ 20
ACRYLAMIDE PERCENTAGE............................................................................................. 20
BLURRY, FUZZY OR SMEARED BANDS / SUDDEN CHANGE IN QUALITY OF GELS
................................................................................................................................................... 21
BUFFER RE-USE .................................................................................................................... 21
Detection issues / DGGE SENSITIVITY................................................................................. 22
DGGE ANALYSIS OF CLONES............................................................................................ 22
GC CLAMPS............................................................................................................................ 22
HIGH DIVERSITY ENVIRONMENTS – MEASURING DIVERSITY................................ 23
MIGRATION OF BANDS INTO THE GEL IS LIMITED OR ABSENT? (NO VISIBLE
BANDS IS A FREQUENT COMPLAINT)............................................................................. 23
MULTIPLE BANDING FROM SINGLE POPULATIONS AND MULTIPLE SEQUENCES
FROM SINGLE POPULATIONS............................................................................................ 24
NON-RIBOSOMAL RNA GENE ANALYSES FOR BACTERIAL COMMUNITY
ANALYSIS............................................................................................................................... 24
PCR ISSUES (There’s no end to these…)................................................................................ 24
Chimeras / Heteroduplex formation...................................................................................... 24
Degenerate primers ............................................................................................................... 25
Double bands ........................................................................................................................ 26
PCR Inhibition due to humics and polysaccharides ............................................................. 26
PCR Contamination .............................................................................................................. 27
Primer cleanup ...................................................................................................................... 27
Primer Mistmatches / PCR Bias ........................................................................................... 27
Primer Design ....................................................................................................................... 28
Quantification ....................................................................................................................... 28
Optimization ......................................................................................................................... 28
General PCR articles............................................................................................................. 28
PERPENDICULAR GELS....................................................................................................... 29
POLYMERIZATION ............................................................................................................... 29
SEPARATION OF BANDS..................................................................................................... 29
SINGLE STRANDED DNA .................................................................................................... 30
SMILING GELS....................................................................................................................... 30
STORING GELS AFTER ELECTROPHORESIS................................................................... 30
WELLS – WAVY, POOR QUALITY, ETC............................................................................ 31
DGGE OF HIGH GC PCR FRAGMENTS.............................................................................. 31
FANCY DGGE TECHNIQUES................................................................................................... 31
RECOMMENDED PCR-DGGE PRIMER SETS AND GRADIENT CONDITIONS ............... 32
Stefan J. Green Stefan@stefangreen.com