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A224 Recombinant DNA Technologies
Problem 4 – Ask for More
Protocol

Background:
DNA was isolated from the fluorescing bacteria. For subsequent cloning steps, additional copies of the gene responsible for the fluorescence are needed. Perform the Polymerase Chain Reaction (PCR) and analyze the PCR reactions via agarose gel electrophoresis.

NOTE: Read and all the instructions carefully before starting your experiment.
Facilitators will guide you on the use of PCR machine and agarose gel electrophoresis.
Be cautious when there is a need to handle items/equipments and the hazardous reagent, Ethidium Bromide (EB) in the EB room.

Materials:

Agarose gel electrophoresis set
DNA ladder (marker)
DNA loading dye
DNA samples (labelled as DNA 1 and DNA 2; DNA 2 is obtained from P3)
Deionised water
Ice in tub
Biohazard bin
Disposable pipette tips
Gloves
Microcentrifuge (table top)
Micropipettes (P1000, P100, P20)
Microcentrifuge tubes (1.5ml)
Paper Towels
PCR machine
PCR master mix (Taq Polymerase, MgCl2, dNTPs, buffer)
Primers (labelled as Primer 1 and Primer 2)

Methods:

LAB 1: AM session (9.15am to 12noon)

Part A: The Polymerase Chain Reaction (PCR)
As a team, you will be using TWO DNA samples to perform TWO PCR reactions; DNA 1 will be given and DNA 2 which is obtained from your P3.

NOTE: If you have TWO DNA samples from P3, choose the ONE with better purity or quantity.

1. Refer to Table 1 and complete the components and volumes needed to make PCR master mix. 2. For TWO PCR reactions, you need to prepare PCR master mix for 2.5 reactions (on ice). Pipette the solutions 1, 2, 3, 4 into a sterile microcentrifuge tube. What is the reason for preparing volumes for 2.5 reactions of PCR master mix solutions? To save time and minimize error in the repeating pipetting steps for small volumes of the same solutions.

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