PCR Detection of Genetically Modified Foods: DNA Extraction, Part II and…
PCR Detection of Genetically Modified Foods: DNA Extraction, Part II and PCR
Wear gloves and use aerosol-barrier tips for the pipettes. Then thaw samples on ice.
Completion of Dry Food DNA Extraction
Add 300 uL of each DNA sample to appropriate place
Reattach plunger and push plunger all the way down.Some waste will be expelled here.
Add 1 mL of Wizard resin to the opening at the top of the syringe
Then add 2 mL 80% isopropanol and reattach plunger and push it all the way.
Set up minicolumns for the samples. Use the syringes and Wizard columns and then label each assembly with the name of the sample that it will contain.
Behead as many 1.5 mL tubes as you have columns.
Label tube with nuclease=free water with initials and place it in warm water bath with lid lock.
Remove syringe barrel from first minicolumn and transfer the minicolumn to open 1.5 mL microcentrifuge tube.
Centrifuge all of these samples.
Transfer this to a new labeled tube and add nuclease free water.
Pull DNA through column by centrifugation again. Save these tubes and place them on ice.
Completion of Wet Food DNA Extraction
Incubate tubes in a boiling water batch for 30 seconds.
Put tubes to ice.
Pipet NaOH into each tube and change tips between each sample.
Add HCl and Tris buffer ot each tube.
Put tube back on ice as soon as you're done.
Boil tubes for 2 minutes and add nuclease free water.
Open the tube and use a sterile stick to mash the contents
PCR (DNA Amplification)
Add 5 uL of DNA to each tube. Change pipet tips between samples.
Add 2 uL of primer mix to the tube.
Label the tubes correctly based off of what they tell you and your specific sample.
Add 18 uL of nuclease free water. Cap and vortex and centrifuge.
Pour each bead from its .5 mL tube into a .2 mL tube
Take samples to TA to place in thermocycler.
Obtain PCR bead and .5 mL tube for each sample being testing including a new negative control.