Lecture 23

Question 1

How to analyze a plasmid

Answer 1

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

Question 2

LB Broth with Antibiotic

Answer 2

• provide continual pressure to maintain plasmid

- ampicillin, kanamycin, tetracycline, chloramphenicol

Question 3

plasmid isolation

Answer 3

• 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

Question 4

If using NaOH to lyse cells….

Answer 4

acidic potassium acetate is needed to treat solution to precipitate debris

Question 5

common modifications of plasmid isolation

Answer 5

• 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

Question 6

PCR

Answer 6

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

Question 7

PCR basic components

Answer 7

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

Question 8

plasmid isolation explain like I’m 5

Answer 8

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