FISH

Question 1

Alpha Satellite Probes- Centromere

Answer 1

Alpha satellite DNA is repetitive DNA sequences located at the centromere

Composed of 171 base pair repeats

Question 2

Dual color/ dual fusion

Answer 2

Signal is 2F1R1G

Question 3

Break apart

Answer 3

Abnormal pattern is
1F1R1G

Normal
2 Fusion

Question 4

Pretreat

Answer 4

To get rid of cytoplasm and debris that can interfere with hybridization.

Slides should be kept at room temp

Slides less than 2 weeks old should be aged in 2XSSC @37 for 30 minutes

Slides more than two weeks old do not need to be aged

Question 5

Denaturation

Answer 5

72C for two minutes in 70% Formamide/2XSSC

Question 6

Satelittle/painting probe denaturation

Answer 6

72C for 5 minutes

Question 7

Postwash

Answer 7

Slides are washed for 2 minutes at 72C

Washed in order to remove unbound probe and probe that has cross-hybridized to nonspecific regions

Question 8

Increase stringency

Answer 8

Increase temp
Increase %formamide
Decrease %salt

Question 9

Fish probs for centromere

Answer 9

CEP

DXZ#

X-chromosome number