PBC-xxxx L Genetics Lab
FALL 2009
Department of Biological Sciences
Florida International University
Brassica rapa as a model for human genetics – Paternity Exclusion
Part 4: DNA Extraction from Plant Material using FTA® Paper and
amplification of molecular markers Na12-H09 and Ra2-E07
I. Objective: The students will learn how to extract DNA from plant tissue by using FTA® cards
(Whatman). In addition they will amplify two molecular markers by performing polymerase chain
reaction.
II. General safety requirement
Always wear labcoat and gloves.
III. Essentials for DNA extraction
(A) Reagents

1 x PBS solution

FTA wash reagent (Whatman biosciences)

1 x TE buffer
(B) Materials

1.5 ml tubes

small pestles

Punches for FTA paper
(C) Equipment: Centrifuge
IV. Essentials for PCR
(A)Reagents

AmpliTaq Gold Polymerase and 10x buffer

MgCl2

Na12-H09 and Ra2-E07 primers

dNTPs 2.5mM

BSA

DEPC water
(B) Materials

0.2 ml PCR tubes

0.1-10µl pipette tips

PCR tube racks

(C) Equipment

Thermal cycler
V. Procedure: Plant DNA extraction
1. Cut the leaf up in small pieces and place in a 1.5 ml tube with 200-300µl of 1x PBS. Use the
disposable pestles to slowly crush the leaf material in the tube. Carefully pipette out
approximately 100-150 µl of the slurried tissue and place on the FTA paper.
2. Dry the FTA paper at room temperature. If using immediately, dry 15-30 minutes. If archiving
dry over night.
3. Punch out 1-3 2mm punches.
4. Place 1-3 paper pieces into 1.5 ml microfuge tube.
5. Add 500 µl of FTA reagent to the FTA paper pieces in the tube. Vortex or shake gently by
hand occasionally for 5 minutes at room temperature. The paper pieces must get wet. Do not
shake too hard or the FTA reagent will all turn to suds.
6. Discard the used FTA reagent by pipetting off into a waste container using a pipette. If there
are suds, you can get rid of them by centrifuging. Cetrifuge the tube for a second or two and
then try to get more liquid off. Try to remove as much liquid as possible. The used FTA
reagent should become quite green.
7. repeat step 5 and 6
8. Add 1 ml TE buffer (or sterile water) to the tube and vortex to suspend the paper pieces. After
the pieces are suspended, let them sit for about half a minute and then shake again. Remove
and discard as much liquid as possible, using the same methods as above. Repeat twice (for a
total of three TE washes). The FTA reagent contains detergent that might inhibit the PCR so it
is important to rinse the paper thoroughly with TE to get rid of the detergent.
9. Dump the rinsed pieces of FTA card onto three layers of Kimwipe and spread them out. Air
dry at room temperature overnight (or 15-30 min).
The rinsed and dried FTA disc is ready to be used for PCR.
VI. Procedure: Polymerase Chain Reaction (PCR)
1. Your TA has repared a mastermix for each of the two primer pairs. The mastermix contains
the following reagents per reaction:
Reagent
Volume of reagent / Reaction, [µl]
DEPC water
14.85
MgCl2 25mM
3
dNTPs 2.5 mM
3
BSA 1%
3
Buffer 10x
3
Primer for. 20µM
0.75
Primer rev. 20µM
0.75
AmpliTaq Gold
0.18
Polymerase
2. Place 8 PCR tubes in a rack and label them on the side and top with group name, name of
molecular marker used and plant characterization e.g. alleged father 1 (AF1), alleged father 2
(AF2), mother (M), child (C). As there is not enough space on the tubes for all this information,
you must use shortcuts. For the primer pairs/markers you could use R for Ra2-E07 and N for
Na12-H09, or whatever works best for you. Just make sure to write immediately in your
notebooks the exact meaning of the shortcuts you chose.
3. Pipette 19 ml of PCR Mastermix containing Na12-H09 primers in 4 of the tubes and 19µl of
Mastermix containing Ra2-E07 primers in the remaining four tubes.
4. Place rinsed FTA discs in the respective PCR tube and make sure the disc is covered with
mastermix.
5. Seal the tubes thoroughly and place on ice until ready to place into thermal cycler.
6.
Once the PCR is completed, the pcr products must be run on a 2.5% Nusieve gel for analysis
(see instructions for gel-electrophoresis in “DNA fingerprint from hair”lab).
PCR cycling:
1. 95°C for 10 min (activation of hot start Polymerase)
2. 94°C for 30 sec
3. 61°C for 1min
4. 72°C for 1min
5. Repeat step 2-4 40x
6. 72°C for 10 min
The PCR product will be analyzed on 2.5% NuSieve agarose gel in the following week. For
instructions see “DNA Fingerprint from hair” lab.
Note: Pipette at least 15µl of PCR product in each well.