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Flashcards in Recombination techniques 2 Deck (14):

What is the purpose of PCR?

making a large amounts of DNA without cloning


What are the steps of PCR?

1)heat separate strands(95 celsius) denature strands
2) annealing -hybridization of primers on both strands( forward and reverse primers) 35-45 Celsius
3)extension- add DNA polymerase and Nucleotides and extend at optimal temperatures


What type of polymerase is used for PCR and why?

TACT - are used in bacteria and operate at an optimal 110 degrees celsius
these poly are used because the denaturing stage goes up to 95 degrees celsius


How do you calculate the number of DNA double strands synthesized in PCR?

n= number of cycles it undergoes
because each round doubles the amount of DNA


What type of nucleotide is used in DNA sequencing?

dideoxyribonucleoside triphosphate


What is a dideoxyribonucleoside triphosphate?

it is missing a hydroxyl group at the 2` and 3`
3` is needed to extend DNA chain


How is DNA sequencing determined?

incorporating a dideoxynucleotide of all four nucleotides will give you different length of the nucleotide. the DNA will be truncated at one base each the heaviest nucleotide will give you the first nucleotide in the sequence and so on


How is the gel read in DNA sequencing?

5` at the bottom
3` at the top


How is the biological relevance of a gene determined?

by mutating that gene and seeing what type of phenotype is produced


What are the strategies for mutating a gene?

gene replacement
gene knockout
gene addition


What is gene replacement?

only the mutant gene is active


What is gene knockout?

no gene is active


what is gene addition?

both normal and mutant gene are active?


How do you knockout a gene?

1)introduce an altered version of the gene into many cells
2)let cells proliferate
3) test for rare colony in which DNA fragment has replaced the copy of normal gene
4)inject cells into embryo
5) introduce embryo into psuedopregnant mouse
60 after birth test offspring for gene

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