26. Characterising genomic or cDNA libraries

Question 1

what are the four techniques to characterise a clone?

Answer 1

1. southern blotting
2. restriction mapping
3. DNA sub-cloning
4. DNA sequencing

Question 2

what does southern blotting do?

Answer 2

identifies specific sequences of DNA. fragments are spereated on a gel, then are transferred to a second medium where hybridisation can occur of the gene of interest

Question 3

what are the 7 steps of southern blotting?

Answer 3

1. digest DNA with restriction enzymes
2. gel electrophoresis
3. denature DNA with NaOH
4. was and neutralise using a buffer
5. transfer DNA to a membrane
6. use a radioactively labelled probe to hybridies the gene of interest
7. use autoradiography to detect probes (ie gene of interest

Question 4

what three things does southern blotting show us?

Answer 4

1. confirms that the DNA has a sequence for the gene of interest
2. find out how big the gene is
3. a good initial screening step

Question 5

what are two limitations of southern blotting?

Answer 5

1. insert can be to large - to much information
2. easier to work with smaller fragments

Question 6

what is sub cloning?

Answer 6

sub cloning cuts inserts into smaller pieces so its easier to deal with. takes fragments out of a vector and ligates it to another vector to make life better

Question 7

what are three benefits of sub cloning?

Answer 7

1. isolates the gene of interest from unwanted sequences
2. facilitates DNA sequence analysis
3. can insert the gene into a more useful vector for proteins