Lecture 23 Flashcards

1
Q

How to analyze a plasmid

A

more bacteria are needed than one colony on a plate can provide so extract single colonies in LB Broth w antibiotic

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2
Q

LB Broth with Antibiotic

A
  • provide continual pressure to maintain plasmid

- ampicillin, kanamycin, tetracycline, chloramphenicol

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3
Q

plasmid isolation

A
  • O/N culture pelleted to remove broth
  • cells resuspended (Tris, EDTA, glucose or salt)
  • cells are lysed (NaOH, SDS, lysozyme, boiling)
  • solution is treated to precipitate debris
  • spin out debris, plasmids remain in supernatant
  • salt added to supernatant to act as a competitor for water (NaCl, LiCl, NaAc)
  • 1-3 volumes of alcohol added to solution to displace water and make DNA less soluble
  • chill on ice to lower solubility
  • spin in centrifuge to collect DNA
  • wash with 70% ethanol to desalt
  • dry and resuspend in water or TE
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4
Q

If using NaOH to lyse cells….

A

acidic potassium acetate is needed to treat solution to precipitate debris

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5
Q

common modifications of plasmid isolation

A
  • resuspend and lyse in one step
  • addition of lysozyme to lysis buffer for gram +
  • remove contaminating proteins using phenol/chloroform extraction
  • resuspension of plasmid in buffer containing RNAse A (digest tRNA, rRNA, mRNA
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6
Q

PCR

A

Using a heat-stable enzyme and short fragments of DNA as start sites we can make billions of copies of a DNA fragment from a single copy

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7
Q

PCR basic components

A
  • template DNA
  • oligonucleotide primers
  • dNTPs
  • heat stable DNA polymerase (Taq)
  • suitable buffer
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8
Q

plasmid isolation explain like I’m 5

A

Cells are pelleted, resuspended, lysed, proteins & genomic DNA precipitated (and discarded), plasmid precipitated, washed, resuspended

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