Lab 1: Plasmid Extraction and Quantification Flashcards
What wavelength does DNA absorb light at?
260nm
What is subcloning?
Putting DNA from a donor plasmid into a recipient plasmid to create a recombinant plasmid
What does the A260/A280 ratio tell us about a DNA sample? What is the ratio for a sample of pure DNA?
How pure it is. 1.8
What happens to the A260/A280 ratio if a sample is contaminated with proteins or aromatic compounds?
It will be <1.8, since proteins and aromatic compounds absorb in the 280 nm range
What happens to the A260/A280 ratio if a sample is contaminated with RNA?
It will be >2, since RNA absorbs in the 260 nm range
What process did we use to isolate the pGFPuv and pHSG298 plasmids from E. coli?
Miniprep
What are the 6 buffers used in the miniprep?
P1, P2, EB, N3, PB and PE
What does the P1 buffer used in the miniprep do? What are some things that are in it? What is the purpose of adding it?
The resuspension buffer, contains salts and RNase. Resuspends the bacterial cells in the pellet
What does the P2 buffer used in the miniprep do? What are some things that are in it? What is the purpose of adding it?
Lysis buffer, contains detergents. Denatures all the DNA
What does the N3 buffer used in the miniprep do? What is the purpose of adding it?
Neutralization buffer. It causes the plasmid DNA to reanneal, but the chromosomal DNA can’t because it is too big
What does the PB buffer used in the miniprep do? What is the purpose of adding it?
For washing, gets rid of salts and contaminants
What does the PE buffer used in the miniprep do? What are some things that are in it? What is the purpose of adding it?
The precipitation buffer. Contains ethanol and precipitates the DNA onto the membrane
What does the EB buffer used in the miniprep do? What is the purpose of adding it?
Elution buffer. Gets the precipitated DNA off the membrane
What is the Nanoview?
A spectrophotometer that can quantify a sample with only a very small amount
