22. Analysis of recombinant plasmids & PCR Flashcards
how can an inserts orientation in a plasmid be determined?
3 steps
using a specific enzyme which can cut the insert and the plasmid assymetrically so they can be distinguished from each other
the digestion must be in the vector and the foregin insert
run the plasmids on agarose gel and it will show the band lengths
what is the PCR reaction?
creates large numbers of a specified DNA sequence in vitro
what are the three requirements for the PCR reaction?
- to be catalysed by DNA polymerase in the 5’-3’ direction
- single stranded DNA as a template
- have a primer with a free 3’ OH group
what are the three stopes in the PCR process?
denaturation: high heat denatures the DNA to split the double strands
annealing: reaction is cooled and primers bind to the complementary sequence
extention: DNA pol synthesising complementary strand of DNA
what are two results of PCR?
Amplification of a single DNA sequence
Almost any DNA molecule can be used as a template
what is the melting temperature of G + C?
4 degrees
what is the melting temperature of A + T?
2 degrees
what are the 3 effects of Mg2+ during anneling?
- Mg2+ determine the fidelity of the polymerase
- needed for dNTP incorportation
- stabilises the primer/template duplex
what are the consequences when Mg2+ conc are to high?
theres a chance that DNA pol will miss the template sequence
what are the consequences when Mg2+ conc are to low?
when Mg2+ conc is low, the amplification will be specfic but low yields are made
what is reverse transcription?
when a sequence of DNA is made from a RNA template
why is RT-PCR used in eukaryote cells? 2 points
- eukaryote genes have introns and extrons, which makes cloning DNA hard because DNA is not completly expressed
- genes in eukaryotes are to big to amplify
how is RT-PCR used in eukaryote cells?
it uses a polymerase that uses RNA as a template. then uses cDNA to make double strnads of DNA with taq polymerase. uses gene specifc primers
