cDNA equivalent to 50ng from each patient sample (tumour and non-tumour tissues) was amplified in two sets of 15-μl reaction mixtures, one for MET and the other for L1-MET. Each reaction mixture contained 7.5 μl of HotStar Taq™ DNA Polymerase (Invitrogen®), 0.8 μl of forward primer (10 μM), 0.8 μl of reverse primer (10 μM) (Supplementary Table S1 ) and 2.4 μl of PCR water. PCR was run in a GSI thermal cycler according to the following protocol: HotStar Taq™ DNA Polymerase was activated by incubation for 15 minutes at 95°C, followed by 3-step cycling [denaturation for 30 s at 94°C, annealing for 45 s at 60°C, and extension for 45 s at 72°C] for 35 cycles and a final elongation step for 10 minutes at 72°C. The amplicons were visualized by gel imaging (FireReader Gel Documentation System, UVITEC, Cambridge, UK) after electrophoretic separation (100 V for 40 minutes) on a 2% agarose gel using horizontal gel electrophoresis (Cleaver Scientific, Ltd., UK). Semi-quantitative analysis was performed by digital analysis of gel images using a freely available image analysis software-ImageJ (Rueden et al. 2017 (link)). GUSB was used as a reference gene for semiquantitative assessment of expression.
Fire reader gel documentation system
Manufactured by Uvitec
Sourced in United Kingdom
The Fire Reader gel documentation system is a lab equipment designed for imaging and analyzing DNA, RNA, and protein gels. It captures high-quality images of electrophoresis gels and provides basic analysis tools.
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Lab products found in correlation
3 protocols using fire reader gel documentation system
1
Semi-quantitative MET Expression Analysis
2
Genetic Primer Amplification Protocol
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A total set of 20 genetic primers (14 ISSR and 6 SSR primers; Bioresource Biotech Pvt. Ltd., Pune, India) for PCR amplification was used for amplification. A PCR assay was performed for all microsatellites as well as gene specific markers. Each 20 μl PCR contained 20-30 ng template DNA, 2.5 μl of 10× PCR buffer, 0.5 μl of 15 mM MgCl2, 1 μl of 10 mM dNTPs, 5 pmol of microsatellite and 10 pmol of gene specific primers and 3.0 units of Taq polymerase. According to the melting temperature of each primer, thermal profiles were standardised for each primer (i.e. marker) [14] . The standard annealing temperatures of all microsatellites and gene specific markers are given in Table 3 (A&B) [9, 17] . The PCR-amplified products were separated by electrophoresis in 2% (w/v) agarose gels at 80 V. The gels were stained with 10 mg ml -1 ethidium bromide and visualised under UV light using a Fire Reader gel documentation system (V10, Uvitec Ltd., Cambridge, UK) and the data were stored for further analysis.
3
ISSR-PCR Amplification Protocol
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15 ISSR primers composed wholly of defined, short tandem repeat sequences with anchor, and representing different microsatellites (di and tri-repeats) have been used as generic primers in PCR amplification of inter simple sequence repeat regions as per the method of Adawy et al., (2002) .A PCR protocol was standardised for all ISSR markers. Each 20 μl PCR contained 25 ng template DNA, 2.5 μl of 10× PCR buffer, 0.5 μlof 15 mM MgCl2, 1 μl of 10 mMdNTPs (Bangalore Genei Pvt. Ltd., Bangalore, India), 10 pmol of each ISSR (BioresourcenBiotech Pvt. Ltd., Pune, India) and 3.0 units of Taq polymerase (Bangalore Genei Pvt. Ltd.). Thermal profiles were standardised for each ISSR primer pair (i.e. marker) based on its melting temperature using a Eppendorf, Master cycler gradient supplied by Eppendorf gradient, 2231, Hamburg Germany was used for cyclic amplification of DNA. The standard annealing temperatures of all ISSR primers are given in Table 2.
The PCR-amplified products were separated by electrophoresis in 2% (w/v) agarose gels at 80 V. The gels were stained with 10 mg ml-1 ethidium bromide and visualised under UV light using a Fire Reader gel documentation system (Uvitec Ltd., Cambridge, UK) and the data were stored for further analysis.
The PCR-amplified products were separated by electrophoresis in 2% (w/v) agarose gels at 80 V. The gels were stained with 10 mg ml-1 ethidium bromide and visualised under UV light using a Fire Reader gel documentation system (Uvitec Ltd., Cambridge, UK) and the data were stored for further analysis.
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