lab2

Question 1

physical mapping is often the quickest way by which one can

Answer 1

verify the size and the oreintation of DNA inserted into a plasmid

Question 2

Southern analysis is often employed to determine

Answer 2

the migration and approximate length of DNA restriction fragments that are similar or identical to that of a known probe

Question 3

Southern blot steps
Target DNA is cut into _______ by _______
and the resulting fragments are
the cleaved, resolve restriciton fragments are _____
then ______ (using _____________)
so as to introduce ________________ to facilitate __________
The DNA is then _____________ usually by _______ under conditions where _____________

Answer 3

restriction fragments

• the action of one or more restriction enzymes
• resolved accosding to size using gel electrophoresis
• subsequently partially depurinated(at acid pH)
• denatured
• strong alkaline conditions
• limited strand breakage
• subsequent steps
• immobilized to a solid support
• capillary blotting
• the membrane binds and retains denatured DNA (high ionic strenth)

Question 4

when blotting is complete, the membrane effectively contains

Answer 4

a ‘mirror image’ of the gel and it’s resolved restriction fragments

Question 5

HOw are denatured immobilized DNA fragments fixed to the membrane by

Answer 5

by UV-crosslinking or baking of the membrane

Question 6

Denatured immobilized DNA fragments are _____________ and exposed in solution to _______ that is labelled in some manner (usually by ________)

Answer 6

fixed to the membrane
denatured DNA of a known sequence
incorporation in vitro of radioactive isotopes such as 32P, 33P or 35S

Question 7

The denatured probe in solution and ‘target’ DNA are incubated under conditions of _____________ where the prove specifically ________________ on the membrane

Answer 7

ionic strength and temperature

hybridizes to its complimentary sequence

Question 8

Following a period of incubation (to allow for ___________) the excess (___________) probe is ________________

Answer 8

• binding and incubation
• unbound
• washed away

Question 9

a probe for the GFP gene allows for ___________ of target DNA

Answer 9

non-radioactive detetion

Question 10

linear double-stranded fragments of DNA migrate in an agarose gel at a rate that is ____________

Answer 10

inversely proportional to the log10 of their size