NA Hybridization

Question 1

General method in NA hybridization

Answer 1

1. blotting
2. probe labeling
3. hybridization
4. washing
5. detection/visualization

Question 2

Basic parts of a Southern blot

Answer 2

weight
paper towels
membrane
gel
sponge
buffer
tray

Question 3

basic parts of a Northern blot

Answer 3

weight
paper towels
membrane
gel
filter paper wick
support
buffer
tray

Question 4

4 ways to label probes

Answer 4

1. nick translation method
2. random primer labeling
3. end labeling
4. PCR labeling

Question 5

general mechanism of the nick translation method

Answer 5

production of nicks by DNase I
excision and replacement with labeled dNTPs by DNA pol I

Question 6

general mechanism of the random primer labeling

Answer 6

denaturation of the DNA
annealing of random hexameric primers
elongation by the Klenow fragment with the labeled dTNPs
release by boiling

Question 7

subunit of DNA pol with the polymerase activity

Answer 7

Klenow fragment/Klenow DNA polymerase

Question 8

enzyme used in end labeling

Answer 8

polynucleotide kinase

Question 9

detection of radiolabeled probes

Answer 9

autoradiography

Question 10

detection of non-radioactively labeled probes

Answer 10

1. colorimetric
2. fluorescence
3. chemiluminescence

Question 11

recognition of digoxygenin by ______ to produce the color _______

Answer 11

1. alkaline phosphatase with antibody
2. purple

Question 12

recognition of biotin by f-tagged ________ to produce fluorescence

Answer 12

avidin

Question 13

recognition of digoxygenin by _________ with antibody to produce chemiluminescence

Answer 13

HRP (horse radish peroxidase)

Question 14

general steps: colony blotting and hybridization

Answer 14

1. you place a membrane on top of a plate containing the colonies
2. replica plate and set aside the master plate (original plate)
3. colonies from the replica plate are lysed and blotted
4. blot is then hybridized with labeled probe and washed
5. detection of the hybridized colonies
6. identification of the target colony from the master plate
7. allow the target colony to grow in liquid culture