Double Stranded Rna Synthesis Lab Report
The objective of these labs were to use sophisticated techniques to produce double-stranded RNA that was incubated with live Drosophila cells to inhibit the expression of our two genes of interest. The overall process of the four labs was to isolate and amplify DNA using the polymerase chain reaction with primers that contained gene-specific sequences to Thread or Dynamin-related protein 1 (drp-1), along with T7 promoter site sequences. The amplified DNA was purified and both strands of the DNA were used as templates for in vitro transcription reactions, producing complementary RNA strands for each of the two genes. The RNA was heated, to separate the RNA hybrids and then the complementary strands were allowed to anneal, forming double-stranded RNA molecules. The purified double-stranded RNA produced in lab was then used with THREAD or drp-1 to cause RNA interference in Drosophila cells. Finally, we examined these cells and photographed them using inverted microscopes.
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This allows Helicase to enter and break hydrogen bonds in the 5’ to 3’ direction. Single stranded binding proteins hold the DNA in place while DNA polymerase adds RNA primer, and DNA polymerase III synthesizes 5’ to 3’. In the lab we manipulated this process by denaturing the hydrogen bonds in a solution heated up to 95 °C. We then annealed the primer by placing the DNA in a solution to 45- 65 °C. This allowed DNA primers to bind to the template strand. Finally, we heated the solution to 72 °C to allow DNA polymerase to replicate a new complimentary strand to the
This allows Helicase to enter and break hydrogen bonds in the 5’ to 3’ direction. Single stranded binding proteins hold the DNA in place while DNA polymerase adds RNA primer, and DNA polymerase III synthesizes 5’ to 3’. In the lab we manipulated this process by denaturing the hydrogen bonds in a solution heated up to 95 °C. We then annealed the primer by placing the DNA in a solution to 45- 65 °C. This allowed DNA primers to bind to the template strand. Finally, we heated the solution to 72 °C to allow DNA polymerase to replicate a new complimentary strand to the