( ions in 1:10 diluted RTplusPCR Buffer a...)cMaster RTplusPCR System for increased sensitivity and product length ,,, in RT-PCR

ions in 1:10 diluted RTplusPCR Buffer at 2.5 mM final Magnesium concentration.

Set-up for One Step RT-PCR reactions



Cycling program parameters



Experiment 2: One Step, Long Range

Target for RT-PCR amplification: human Tuberous Sclerosis Factor (hTSF) mRNA, a 5.3 kb fragment. Template RNA: total RNA purified from human HELA cells, template concentrations in the reactions are 100 and 10 ng.
Protocol: Eppendorf cMaster RTplusPCR Kit, special One Step
RT-PCR protocol for long templates.

Set-up for One Step RT-PCR reactions



Cycling program parameters



Experiment 3: Two Step RT-PCR

Targets for Two Step RT-PCR amplification:

Alpha Tubulin mRNA (mouse and human), 500 bp fragment
Tumor Necrosis Factor Receptor (TNFR1) mRNA (mouse and human), 1.3 kb fragment
Human Tuberous Sclerosis Factor (hTSF) mRNA, 5.3 kb fragment
Mouse Dynein mRNA, 12.3 kb fragment
Protocol: Eppendorf cMaster RTplusPCR Kit, Two Step RT-PCR
protocol for standard sized and long templates (manual). All reverse
transcription reactions were primed with 0.5 g Oligo(dT)20 primer.


Set-up for the first step RT reaction



Set-up for the second step PCR reaction



Set-up for RT reaction



Two step cycling program used for Tubulin and TNFR1

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