Sanger Sequencing

Question 1

Can you see repeats through putting pcr products on electrophoresis gel?

Answer 1

no, it’ll show the same size bands

Question 2

What does sanger sequencing require

Answer 2

template, polymerase, one primer, ddNTPS, and dNTPS

Question 3

Does sanger sequencing amplify the product?

Answer 3

no!

Question 4

Why do we use ddNTP in sanger sequencing?

Answer 4

because its missing an OH, so it can be added to a seuqnces but has to terminate after added.

Question 5

How does sanger sequencing work?

Answer 5

sam steps as pcr, except you do a bunch of diff reactions and put in a primer in each, polymerase, template, dntps, and ddntps ( so in one tube datp and ddatp)

Question 6

At what ration do you add ddntps and dntps?

Answer 6

300 dntps to 1 ddntp

Question 7

What does sanger sequencing result in?

Answer 7

DNA fragments of diff sizes in one reaction tube, as ddntp will stop the additions at diff points in time

Question 8

How do you sequence the product in the rxn tubes?

Answer 8

add it to gel (polyacrimide as it differntittae sbetwene 1 bp differences), add each rxn to it’s own well and see sizes

Question 9

What is the automated method of sanger sequencing?

Answer 9

all ddntps are in the same rxn tube and coloured differently by flourescent markers they are then seperated by capillary electrophoresis and detected by a laser beam