Lb L4

Question 1

3 main steps for PCR

Answer 1

1. Denaturation
2. Annealition
3. Extension

Question 2

Denaturation

Answer 2

95 degrees
Turns double stranded dna into single stranded

Question 3

Annealition

Answer 3

50-67 degrees
Addition of primers using dnpts, dna pol

Question 4

Exyension

Answer 4

67-72 degrees

Question 5

Primers determine what is amplified

Answer 5

Dna pol cant synthesis dna without primer
20-25 nt
Can calculate temp of primers

Question 6

.ddNTPs

Answer 6

Cant extend DNA seq

Question 7

.ddNTPs vs dNTPs

Answer 7

Dd- cant extend has an H group insteasd of OH

Question 8

Sager sequence

Answer 8

Radioactive primer in 4 seperate reax with flurocent dye - tract which ddNTP was added
Gel elecvtrophprosios seperate on size and charge

Question 9

Next generation sequencing

Answer 9

Shearing of dna — small frags
Ligate adaptor
Add probes
Probes bind to dna, non bound dna washed away
Flow cells containing complementary adaptors
Pcr using adaptors to generate cluster
Clusters needed so fluroscent signal is strong enough to be detected by camedra

Sheraing of dna—ligate adaptors—probes(magnet) —- flow cell contain complementary adaptor— pcr— clusters— detects fluroscent by caamera

Question 10

What are probes

Answer 10

Complemantary sequence + tag (eg biotin)