Recovering cDNA bands from differential display RT-PCR gels using a transparency film mask.
We describe here the use of a transparency film as a mask for recovering bands from differential display reverse transcription-polymerase chain reaction (DDRT-PCR) gels. This method represents a simple and rapid way to isolate the differentially expressed bands from dried and nondried polyacrylamide, radioactive and nonradioactive, denaturing and native DDRT gels. A transparency film is overlaid on the DDRT autoradiogram, and the marks and bands of interest are drawn using a permanent marker. The reproduced band marks are cut out of the transparency sheet with a scalpel to facilitate the recovery of the desired bands. The transparency film mask is then overlaid on the top of the gel and the bands are recovered from the gel. The use of the transparency film mask avoids damage to the autoradiogram and is also extremely useful in DDRT-PCR experiments involving different RNA samples that produce band patterns of different intensities that require many X-ray exposures for different periods of time.
de Oliveira
J G
JG
Departamento de Microbiologia, Universidade Federal de Minas Gerais, Brasil.
Ferreira
P C
PC
Kroon
E G
EG
D001345:Autoradiography; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000032:analysis* / Q000302:isolation & purification*; D003626:Data Display; D004591:Electrophoresis, Polyacrylamide Gel; D004867:Equipment Design; D005347:Fibroblasts; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*
D018076:DNA, Complementary