Your nice, shiny new molecular laboratory was a thing of beauty and cleanliness--until you took your first set of PCR reactions out of the thermocycler
, and opened the caps.
RT-PCR was conducted by using a GeneAMP PCR System 9700 thermocycler
(Applied Biosystems, Foster City, CA, USA) with the following thermocycling profile: 30 min at 42[degrees]C; 15 min at 95[degrees]C; 35 cycles of 30 s at 94[degrees]C, 30 s at 42[degrees]C, and 45 s at 72[degrees]C; followed by a final 7-min extension at 72[degrees]C.
Because the critical denaturation temperature needs to be controlled precisely during PCR, a thermocycler
with a high degree of accuracy and precision in temperature control is desirable, i.
Accomplishments towards this purpose included: (1) Designing the promoter and fluorescent protein (FP) nucleotides that should be responsive to MSG; (2) Assembling and amplifying the promoter and the FP genes using the nucleotides in a polymerase chain reaction (PCR) thermocycler
instrument; (3) Transforming the promoter and the FP into a bacterial plasmid vector; (4) Testing the completed E.
Amplifications were performed on a DNA thermocycler
(Perkin-Elmer) with the following program: an initial incubation at 97[degrees]C for 6 min, followed by 35 cycles of 94[degrees]C (1 min), 58 [degrees]C (1 min) and 72[degrees]C (2 min), and a final extension at 72[degrees]C for 7 min.
0 [micro]l/reaction was placed in costar 96 micro-well PCR plates, spinned briefly in a micro centrifuge before it was run using the DNA PCR thermocycler
High school teachers might also be able to borrow more expensive equipment, such as a thermocycler
or UV-gelbox from a neighboring community college or university, if they want to perform this exercise on their own.
Upon completion, the reaction tubes were held in the thermocycler
at 4[degrees]C until removed for electrophoresis.
3,4) Once the nucleic acids are purified, they are transferred to either 96-well or 384-well plates containing the master mix (which was manually prepared) and placed in the thermocycler
where the samples are subjected to real-time PCR.
Genotyping for Ser49Gly and Arg389Gly was carried out by Real Time Thermocycler
(ABI Prism 7700, Fosters city, CA, USA) using Taqman SNP genotyping assay method which employs the fluorogenic 5'nuclease chemistry (also known as Taqman probe-based chemistry) to enable detection of specific PCR product.
1999) and consisted of 2 min at 95 [degrees]C, followed by 27 cycles of 1 min at 95 [degrees]C and 1 min at 56 [degrees]C (Ts1 and Ts4), 54 [degrees]C (Ts2), or 57 [degrees]C (Ts3), and 1 min at 72 [degrees]C on a T-Gradient thermocycler
(Biometra, Labrepco Inc.
The tubes are then placed into a device called a thermocycler