Ch. 6 Study Questions

Question 1

What is the purpose of denaturation of ds target DNA after electrophoresis and prior to transfer in Southern blot?

Answer 1

dsDNA is denatured to allow hybridization of the probe. ssDNA also binds to nitrocellulose filters.

Question 2

Name two ways to permanently bind nucleic acid to nitrocellulose following transfer.

Answer 2

1. Bake the filter at 80C for 30 min

2. Upon exposure to UV light, nucleic acid will be cross-linked to nitrocellulose

Question 3

If a probe for S. blot was dissolved in a hybridization buffer that contained 50% formamide, is the stringency of hybridization higher or lower than if there were no formamide?

Answer 3

The stringency is higher, since formamide facilitates denaturation of the dsDNA

Question 4

If a high conc. of NaCl were added to a hybridization solution, how would the stringency be affected?

Answer 4

Stringency would be decreased since the high salt conc. exclude nucleic acids, forcing them close together and promoting hybridization.

Question 5

Does and increase in temperature from 65C to 75C during hybridization raise or lower stringency?

Answer 5

Raises stringency. Heat promotes separation of the H bonds holding the two ss of dsDNA together

Question 6

At the end of the S. blot procedure, what would the autoradiogram show if the stringency was too high?

Answer 6

Faint or no bands

Question 7

A N. blot is performed on an RNA transcript with a sequence. The probe sequence is identical. Will this probe hybridize to the target transcript?

Answer 7

No, their sequences are identical and not complementary.