PCR Testing of GM Food
PCR for Testing for GM Food
Credits: Susie Stevens of Latta High School, Oklahoma; reported protocol derived from DNA Learning Center of Cold Spring Harbor Laboratory.
Part A. Isolation of DNA from Food Samples or Plants: Method One
Materials and Equipment:
PrepMan™ Ultra Sample Preparation Buffer (#4322547 from Applied Biosystems)
Sterile micropestles
Boiling water bath
Microcentrifuge
Isopropanol
Distilled water
3M potassium acetate
Micropipettor with tips
Microtubes (1.5 mL)
TE Buffer
Corn food samples or corn plant leaves, Bt+ or Bt-
Procedure:
Purchase food samples or obtain plant samples and assign.
Pulverize food samples that are hard or crunchy.
Place very small amount of food sample in 1.5 mL microtube (less than 100 µl volume).
Add 400 µl of PrepMan Ultra Buffer reagent and use pestle to grind food for one minute.
Vortex 10 seconds.
Put in boiling water bath for 10 minutes; cool 2 minutes at room temperature.
Centrifuge 3 minutes at 16,000 x g.
Transfer 75 µl of supernatant into clean microtube and add 75 µl of distilled water. (Okay to stop here, freeze samples and resume procedure in later class period.)
Add 50 µl of 3M potassium acetate (to precipitate proteins).
Add 155 µl of isopropanol, mix, leave at room temperature for 3 minutes.
Centrifuge at 14,000 x g for 6 minutes; decant supernatant.
Centrifuge again as above and pipette out any remaining alcohol.
Let pellet air dry for 10 minutes.
Resuspend DNA pellet in 100 µl of TE buffer.
STOP POINT. Store DNA template at -20º C.
Part A. Isolation of DNA from Food Samples or Plants: Method Two
Materials and Equipment
Saline solution (0.9%NaCl), 1 mL
10% Chelex, 100 µl
Sterile micropestles
Boiling water bath
Cup of ice
Microcentrifuge
Distilled water
Micropipettor with tips
Microtubes (1.5 mL)
TE Buffer
Procedure:
Purchase food samples or obtain plant samples (1 cm x 1 cm) and assign.
Pulverize food samples that are hard or crunchy.
Place very small amount of food sample in 1.5 mL microtube ( less than 100 µl volume).
Add 1 mL of saline solution and use pestle to grind food or plant for one minute.
Centrifuge 1 minute at highest speed of microcentrifuge (using balanced configuration of tubes).
Carefully pour off supernatant and discard. Be careful not to disturb cell pellet at bottom of test tube. A small amount of saline can remain in the tube.
Resuspend pellet by pipetting in and out (add up to 30 µl saline if needed).
Withdraw 30 µl of cell suspension, and add to tube containing 100 µl of chelex. Shake well to mix.
Put in boiling water bath for 10 minutes; cool 2 minutes on ice.
Shake tube. Centrifuge 1 minute at 16,000 x g.
Transfer 60 µl of supernatant into clean microtube. Avoid cell debris and chelex beads. This is your DNA sample. (Okay to stop here, freeze samples and resume procedure in later class period.)
STOP POINT. Store DNA template at -20º C. or on ice till ready to begin part II.
II. Part B. Amplification of 35S promoter (CaMV) (161 bps) and Bt Cry1A gene (146 bp) by PCR
Materials and Equipment
Thermal cycler
micropipettors and sterile aerosol-barrier tips
Ready-to-go PCR Beads (Amersham Biosciences #27-9558)
Microtubes
Deionized distilled water
Template DNA isolated in Part A from food or plant samples
*Cresol red loading dye: Add 17 g. sucrose to 49 mL of sterile distilled water; dissolve. Add 1 mL of 1% cresol red dye solution. Mix. Store at 4º C. Makes 50 mL.
35S primer mix:
515 ml of distilled water
460 ml of cresol red loading dye*
75 ml of 5' and 3' primers at 15 pmols/ml using following primer sequences:
Forward 5' CCG ACA GTG GTC CCA AAG ATG GAC
Reverse 5' ATA TAG AGG AAG GGT CTT GCG AAG G
Bt primer mix:
515 µl of distilled water
460 µl of cresol red loading dye
75 ml of 5' and 3' primers at 15 pmols/µl using the following primer sequences:
Forward 5' CTG GTG GAC ATC ATC TGG GGC ATC TTC G
Reverse 5' TTG GTA CAG GTT GCT CAG GCC CTC C
(Suggestion: Heat primers one minute at boiling or near-boiling before use from freezer storage)
Procedure:
Each sample would have two PCR reaction tubes, each containing PCR ready-to-go bead: 1 for 35S primer and 1 for Bt primer. Label tubes.
Add 22.5 ml of Bt primer mix to one tube, 35S primer mix to other tube. Tap tube to dissolve bead completely in mix.
Use fresh tip to add 2.5 ml of sample DNA to each reaction tube and mix completely. Pool reagents by quick spin in microcentrifuge or by sharply tapping the tube bottom on the lab table.
Store on ice till ready to put into thermal cycler; move into thermal cycler as quickly as possible.
Use the following program:
15 cycles of: 98ºC for 20 sec, 58oC for 4 sec, 72ºC for 15 sec
15 cycles of: 98ºC for 20 sec, 54oC for 4 sec, 72ºC for 15 sec
15 cycles of: 98ºC for 20 sec, 50oC for 4 sec, 72ºC for 15 sec
Hold: 4ºC
STOP POINT. Store PCR product tubes in -20 degree freezer till ready to run gels.
Part C. Gel Analysis of PCR Results
Materials and Equipment
2% agarose gel made in TBE
TBE buffer
PCR samples from part B
1 mg/mL ethidium bromide
micropipettors and tips
electrophoresis chamber and power supply
gloves
100 bp DNA marker (ladder)
UV transilluminator
Procedure:
Load 15 ml of each PCR sample into wells of 2% agarose gel.
Load 10 ml of 100 bp DNA marker into another well.
Electrophorese at 130 volts for 20-30 minutes.
Stain gel in an ethidium bromide solution (1 mg/mL) for 10-15 minutes.
Observe gel under UV to detect bands. If you are using a 100 bp Ladder, the Bt gene (cryA1) (146 bp) and the 35S promoter gene (161 bp) should appear as a band between the 100 bp and 200 bp fragments of Ladder. Smeary bands below 100 bps in size usually represent primer-dimers.
- See more at: http://www.occc.edu/bbdiscovery/pcrgm-food.html#sthash.txwGjdZa.dpuf