10. In situ Hybridization Flashcards
1
Q
2 types of in situ hybridization methods
A
FISH and CISH
2
Q
Role of formamide in an ISH hybridization buffer and its benefit
A
Lowers the required DNA denaturation temperature (lowers Tm) from 90°C to 65-72°C
3
Q
When is biotin blocking performed?
A
After permeabilization but BEFORE hybridization
4
Q
Why is ISH not affected by tissue DNA degradation but PCR is
A
PCR - primer extension may be stopped due to breakage = no amp
ISH - probes are thousands of bp long so pairing can occur even if DNA is fragmented
5
Q
Purpose of double-baking tissues mounted to slides for ISH
A
- 1st time to adhere section to slide
- 2nd time to denature DNA and obtain primary structure suitable for hybridization
