Molecular techniques to identify mutations in C. elegans - Experiment 6

Question 1

What types of mutations can be seen with PCR and gel electrophoresis?

Answer 1

Indels

Question 2

Why can’t point mutations be seen with PCR and electrophoresis?

Answer 2

They are all the same size

Question 3

When do we use PCR?

Answer 3

To amplify a target sequence of DNA to be able to detect and manipulate it

Question 4

What 4 things are required to make DNA replication occur in vitro?

Answer 4

1. A template strand
2. Primers
3. dNTPs
4. DNA polymerase

Question 5

What 4 things are required to get the chain reaction for PCR started?

Answer 5

1. The DNA template
2. Two different types of primers
3. dNTPs
4. DNA polymerase

Question 6

Why do we need two different types of primers for PCR?

Answer 6

One for each strand, eventually the target sequence will get boxed in between the primers

Question 7

Why do we need a special machine for PCR?

Answer 7

The temperature needed to be changed several times. It needed to be at 94°C to get the DNA strands to separate, then cooled down to 55 to get the primers to pair up with the DNA, then heated up to 72 for optimal function of DNA polymerase

Question 8

What is the minimum number of cycles needed to amplify the target sequence enough?

Answer 8

25

Question 9

What do indels look like on the gel?

Answer 9

Insertions are larger than WT, so aren’t as far. Deletions are smaller than WT, so are farther

Question 10

Why is bromophenol blue added to the sample?

Answer 10

Gives it a visible blue colour that lets us track how much the DNA has moved, it runs like a piece of DNA 300 bp long

Question 11

Why is sucrose added to the sample?

Answer 11

Make it sink to the bottom of the well

Question 12

How are we able to see DNA on the gel?

Answer 12

A fluorescent dye that gets intercalated into the DNA structure is added - either ethidium bromide or Sybr-safe