The primer set used was FF390 (5′-CGA TAA CGA ACG AGA CCT-3′) and FR1 (5′-AIC CAT TCA ATC GGT AIT-3′) with a GC-clamp (5′-CCC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GCC G-3′) at 5′ end56 was used to amplify ~390 bp fungal 18 S rRNA gene fragments of different samples in the coffin chamber. A DCode Universal Mutation Detection System (Bio-Rad, USA) was used for DGGE analysis. Approximate 150–250 ng PCR amplicons from each sample were electrophoresed on a 8% acrylamide- bisacrylamide gel, with 45% to 75% denaturant at 100 V for 10 h in 1×TAE running buffer at 60 °C. The gels were stained for 20 min with SYBR Green I nucleic acid gel stain (1:10000 dilution) (Invitrogen, USA). The gels were visualized and digitalized by using a Gel DocTM EQ imager (Bio-Rad, USA) combined with Quantity one 4.62 (Bio-Rad, USA). The representative bands were excised, left overnight in 25 μl Milli-Q water, reamplified and run again on the DGGE system to ensure purity and correct mobility of the excised DGGE bands. Correct PCR products were purified using the QIAquick PCR Purification kit (QIAGEN) before cloning.
Gel doctm eq imager
Manufactured by Bio-Rad
Sourced in United States
The Gel Doc™ EQ Imager is a compact and versatile imaging system designed for analyzing DNA, RNA, and protein gels. It captures high-quality images of gel-based samples using a charge-coupled device (CCD) camera and adjustable lighting. The system is capable of imaging a variety of stained gels, including agarose, polyacrylamide, and SDS-PAGE gels.
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Lab products found in correlation
2 protocols using gel doctm eq imager
1
Fungal Community Profiling via DGGE
2
Bacilli Ribosomal Gene Profiling via DGGE
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A DCode Universal Mutation Detection System (Bio-Rad, Hercules, Calif.) was used for DGGE analysis. Approximate 150-250 ng PCR amplicons of 350-bp Bacilli ribosomal genes from each soil sample were electrophoresed on a 10% acrylamide-bisacrylamide gel, with 30% to 70% denaturant at 130V for 8 h in 1×TAE running buffer at 60°C. The gels were visualized and digitalized by using a Gel Doc TM EQ imager combined with Quantity one 4.4.0 (Bio-Rad). The representative bands were excised, left overnight in 25 μl Milli-Q water, reamplified and run again on the DGGE system to ensure purity and correct mobility of the excised DGGE bands.
Correct PCR products were purified using the QIAquick PCR Purification kit (QIAGEN) before cloning.
The purified PCR amplicons of the excised DGGE bands were cloned into a pMD18-T vector (TaKaRa) and transformed into Escherichia coli DH5α competent cell. Six random clones containing correct gene size for each DGGE band were sequenced by Invitrogen Sequencing Department in Shanghai. DNASTAR software package was used to manually check and compare the clone sequences. One representative clone sequence with high quality after sequence comparison from each band was used for phylogenetic analysis.
Correct PCR products were purified using the QIAquick PCR Purification kit (QIAGEN) before cloning.
The purified PCR amplicons of the excised DGGE bands were cloned into a pMD18-T vector (TaKaRa) and transformed into Escherichia coli DH5α competent cell. Six random clones containing correct gene size for each DGGE band were sequenced by Invitrogen Sequencing Department in Shanghai. DNASTAR software package was used to manually check and compare the clone sequences. One representative clone sequence with high quality after sequence comparison from each band was used for phylogenetic analysis.
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