Basic PCR – Polymerase Chain Reaction
BACKGROUND:
Name _________________________
Sources: R. Aleff, Mayo Clinic; Campbell Biology Text, 8th edition; Wikipedia; www.invitrogen.com; www.macvector.com
PCR: In PCR a small amount of DNA can be amplified. A target segment of DNA can be copied billions of
times in a few hours. PCR uses a 3 step cycle to bring about a chain reaction that produces an exponentially
growing population of identical DNA molecules. One cycle of PCR includes 3 steps. In a classroom setting
20-50 cycles are generally run.
Step
What is happening
Details
Denature
DNA strands are separated.
94-95 ºC for 30 sec to 1 minute.
Anneal
Primers are attached.
50 – 70 ºC depends on the primers
for 1 minute.
Extensions
Complementary nucleotides are added to each single
strand of DNA using a heat resistant DNA Polymerase.
In our lab we are using Taq polymerase.
72 ºC for 1 minute.
pBR322: pBR322 is the cloning vector that we will be using.
(the p stands for plasmid and BR for the creators Bolivar and Rodriguez.) It
is a plasmid and is derived from a strain of E.col. pBR322 has
4361 bp and we will be amplifying the region from 34-688 bp
Primers: are 18-24 base pairs in length and are single stranded
DNA.
Taq Polymerase: catalyzes the incorporation of dNTPs
into DNA
dNTPs: Deoxynucleotides triphosphates - the building blocks
from which the DNA polymerases synthesizes a new DNA
strand.
PROCEDURE: Basic PCR pBR322 Amplification
Source: R.Aleff Mayo clinic
1. Label a 0.2 ml PCR tube with your initials and pBR322
2. Add 23.0ul PCR supermix (contains buffer, DNTP’s, & Taq polymerase)
3. Add 1.0ul pBR322 5-10 ng/ul
4. Add 1.0ul of 5’ & 3’ primer
5. Mix gently
6. Spin down in pico-fuge 2 seconds
7. PCR Set-up
1 cycle
35 cycles
Fill In
Hold
Hot start
Denature
Anneal
Extension
Extension
Cool
95 degrees
95
55
68
68
4
8. Place __15ul PCR & 5 ul track dye into well on E-gel for __30__ minutes
9. Look for band at 654 base pair
10. Primers: 5 ‘ AATGCGGTAGTTTATCACAG
3’ GCTGACTGGGTTGAAGGCTC
4-10 minutes
1 minute
1 minute
1 minute
7 minutes 1 cycle
Infinity