Homework Soursename the reagents that are included in a typical PCR reactio
name the reagents that are included in a typical PCR reaction and give an example of typical PCR run conditions.
name the reagents that are included in a typical PCR reaction and give an example of typical PCR run conditions.
Solution
Polymerase Chain Reaction (PCR) is used to amplify the desired gene which is required in large amounts. Basic reagents required to carry out the reaction are as follows,
1. Desired Gene (Template DNA)
2. Primers (Forward and Reverse)
3. Taq Polymerase (DNA polymerase derived from the bacteria named Thermus aquaticus)
4. Nucleotides (dATP, dGTP, dCTP, dTTP)
5. PCR buffer
Necessary conditions to run PCR:
Denaturation is carried out to denature the double stranded DNA into single stranded DNA. Then Annealing of primers to single stranded DNA and adding nucleotides is the extension step.
Each cycle has denaturation, annealing and extension phase.
95°C - Denaturation
45-68°C - Annealing
68°C - Extension
| Steps | Temperature | Time |
| Initial Denaturation | 95°C | 30 seconds |
| 30 Cycles | 95°C 45-68°C 68°C | 15-30 seconds 15-60 seconds 1 minute/kb |
| Final extension | 68°C | 5 minutes |
| Hold | 4-10°C |
