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Question: in this part of the activity you will go through...

Question details

In this part of the activity, you will go through the process of predicting the outcomes of primer extension

assays.

For each of the 5 experiments below, draw the data for the following primer extension reactionsin the gel onthe next page:

* = radioactive label

Experiments 3 and 4 used dideoxy nucleotides. These nucleotides lack both the 2’ and 3’ hydroxyl grou

ps.

What effect will a dideoxy nucleotide triphosphate have on DNA replication?

Experiment3:

1. dsDNA: 5'-AAGACAATAACACCTGTATAACAAATGGTCGGAGTGCCGCGATGAAACTGCGCAAAATCCT-3'

2. ssDNA: 5'-*AAATGGTCGG-3'

3. dATP, dGTP, dTTP and ddCTP

4. DNA polymerase & buffer

Steps:

a. Mix 1 & 2. Heat to 95 ̊C and let cool.

b. Add 3 & 4. Incubate at 37 ̊C for 30 minutes.

c. Add loading buffer, run on a denaturing page gel.

Experiment 4:

1. dsDNA: 5'-AAGACAATAACACCTGTATAACAAATGGTCGGAGTGCCGCGATGAAACTGCGCAAAATCCT-3'

2. ssDNA:5'-*GGCACTCCGT-3'

3. dNTP and ddATP

(so that [dATP] ~1/10x[dATP])

4. DNA polymerase & buffer

Steps:

a. Mix 1 & 2. Heat to 95 ̊C and let cool.

b. Add 3 & 4. Incubate at 37 ̊C for 30 minutes.

c. Add loading buffer, run on a denaturing page gel.

Experiment 5:

1. dsDNA: 5'-AAGACAATAACACCTGTATAACAAATGGTCGGAGTGCCGCGATGAAACTGCGCAAAATCCT-3'

2. ssDNA: 5'-*AAATGGTCGG-3' & 5'-*GGCACTCCGA-3'

3. dNTP

4. DNA polymerase & buffer

Steps:

a. Mix 1 & 2. Heat to 95 ̊C and let cool.

b. Add 3 & 4. Incubate at 37 ̊C for 30

minutes.

c. Add loading buffer, run on a denaturing

page gel.

- I just need a picture or list of what the bands are and their sizes, and why they cut at the place they do, thank you :).

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