GMO Lab report (1 page) 6 hours ASAP

Biol 390-Lab 13 GMO – PCR

Objective

· Test crops or processed foods for genetic modification

· In this lab DNA will be amplified

Background

· Genetic engineering has been used to improve traits of many crops such as yield, diseases resistance, pesticide tolerance. These crops are generally known as genetically modified organisms or GMOs.

· There are some who want to not consumer GMO crops for various reasons, so being able to detect plants and foods that do not contain GMO is important

· There are two ways to detect GMO

· Use an ELISA assay, which is an antibody-based test, to detect the protein that are being expressed.

· Use PCR that can amplify engineered sequences such as

· 35S promotor from the cauliflower mosaic virus (CaMV 35S)

· NOS (nopaline synthase) terminator from Agrobacterium tumefaciens

The PCR assay we will use primers for both NOS and CaMV 35S (red dye added) as well as primers against plant DNA as a check on the extraction and amplification.

Materials

· Ice bath

· GMO master mix – red

· Plant master mix – green

· GMO positive control

· Test food DNA

· PCR tubes

· PCR adaptors

· Foam micro tube holders

· 2-20ul micropipette

· 200 ul micropipette

· Thermal cycler

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Procedure

Preparation before the lab

· Thaw GMO positive DNA template and pulse spin tubes

· Add 50ul of GMO positive DNA template to 4 screw cap tubes labeled GMO+

· Store on ice

· Thaw master mixes and primers 30 min to 1 hour before the lab

· Keep tubes on ice

· Label 5 screwcap tubes PMM (plant master mix)

· Label 5 screw cap tubes GMM (GMO master mix)

· Add 550ul of master mix to one PPM tube and 550ul to one GMM tube

· Add 11ul of green primers to the master mix in the PMM tube mix and store on ice

· Add 11ul of red primers to the master mix in the GMM tube mix and store on ice

· Aliquot 65ul of the plant master mix PMM to each of the remaining 4 tubes

· Aliquot 65ul of the GMO master mix GMM to each of the remaining 4 tubes

· Each workstation gets a tube of each

Set Up PCR

· Wear Nitrile Gloves

· Label six PCR 1 through 6 and keep the tubes on ice for the remaining steps.

· Using a fresh tip each time add 20ul of the appropriate master mix to each tube (see the above table).

· Using the above table add 20ul of the appropriate DNA to the appropriate tubes. (important - only transfer the supernatant from food extract samples). If the pellet is loose re-centrifuge the sample.

PCR

· Program the thermal cycler as follows:

· After the PCR has been completed the tubes will be frozen at -20oC.