PCR INSTRUCTIONS (Bead Method)
MATERIALS
27F primer
1492R prime
PCR beads
Sterile nuclease-free water
Streak plate of bacterial isolate
PROTOCOL*
1. Obtain PCR bead tubes, which contain Taq polymerase (a heat resistant enzyme)
and other necessary reagents. Label tubes appropriately.
2. Dispense 23 μL of ultra-pure sterile water into the PCR bead tube. The bead will start
to dissolve and slightly effervesce.
3. Add 1 μL of forward primer (27F, 20 μM). As you dispense the primer solution, insert
the micropipette tip into the mix so that the small volume goes directly into the mix.
4. Add 1 μL of reverse primer (1492R, 20 μM) to the mix. This will bring the volume of
the reaction mix to 25 uL.
5. Using a micropipette tip, carefully touch the colony on the streak plate. A small dab
that collects a small yet visible blob of cells will provide enough DNA template for the
reaction. Optional: resuspend cells in 100 uL phosphate-buffer saline (PBS) solution,
mix thoroughly by gently vortexing, and add 5 uL of the cell suspension to the mix. This
will bring the reaction mix volume to 30 uL.
6. Using a micropipetteman, mix the reaction mix by gently swishing the solution up and
down several times. Cap the tubes. If necessary, gently flick or vortex for a few seconds
to attain a more uniform mix.
7. Transfer tubes to thermal cycler (PCR machine).
8. Select appropriate program† to start cycling (about 2 hours).
9. Once cycling is complete, remove tubes and keep in ice. Follow your instructor’s
instructions about storage, and follow up protocols to quality test the PCR products and
prepare them for sequencing.
*(Protocol adapted from “puRe Taq Ready-To-Go PCR Beads” guide)
GEL ELECTROPHORESIS INSTRUCTIONS
MATERIALS
Agarose
TBE buffer
Ethidium bromide
Erlenmeyer flask
Microwave oven
Loading dye
Gel tray, comb, and gel electrophoresis apparatus
UV chamber
PROTOCOL
1. Weigh 1 g of agarose and mix with 100 mL TBE buffer in an Erlenmeyer flask – this
will make a 1% agarose gel
2. Microwave contents of Erlenmeyer flask until boiling (1-2 mins) or until mixture
becomes transparent.
3. Allow mixture to cool down (10-15 mins)
4. Carefully add 10 μL of ethidum bromide to mixture
(Warning: ethidium bromide is a DNA intercalating agent and mutagen. Handle this
compound only in designated areas. Wear gloves and avoid contact with skin).
5. Pour mixture into gel tray with appropriate comb and allow mixture to solidify (about
15 mins).
6. Carefully pull comb off gel. Place gel tray into chamber with TBE buffer and start
loading wells:
a. Load 1 kb ladder into first well.
b. Mix 5 μL of PCR product with 1 μL loading dye and dispense into wells.
7. Plug electrodes on appropriate terminals of the chamber (keep in mind that DNA has
a partial negative charge).
8. Allow gel to run for 40-60 minutes (at 100 milliamps/volt).
9. Finally, carefully remove gel from tray and place in UV chamber to get a photograph
(expect to see continuous band at about 1.7 kb).
16S rRNA Primers:
27F
5’ – AGA GTT TGA TCM TGG CTC AG – 3’
1492R
5’ – GTT ACC TTG TTA CGA CTT – 3’
M = A or C according to standard IUPAC ambiguity codes.
PCR cycling:
94oC for 10 min if doing colony PCR (or else just 1min)
94oC 30 sec
58oC 30 sec
72oC 1 min 50 sec
Cycle 30 times
72oC for 10min