Test genomic DNA for amplification by PCR by setting up 10 µL pilot reactions. Reactions might require Qiagen Q solution for optimal PCR (or they might be inhibited by Q); test by setting up +Q/-Q reactions (see Pilot PCR protocol).
The following example is a preparative PCR experiment for one (1) genomic DNA sample using Qiagen’s Fast Cycling
PCR kit.
20 µL total volume
Genomic template DNA = 1 µL per reaction
MASTER MIX (stock reagents from Qiagen)
2x Fast Cycling Master Mix 10
BSA (1 mg/mL non-acetylated stock)
63F-HEX or 63FD4 labeled primer (5 pmol/µL stock)
1387R primer (10 pmol/µL stock)
Q solution (if Q omitted, substitute with water)
Genomic DNA template
H
2
O
0.4
1.6
0.8
(4.4)
1 to bring total volume to 20 µL
Total Master Mix volume
PCR CONDITIONS
95°C, 5 min initial denaturation & polymerase activation.
35 cycles of 96°C, 5 sec; 55°C, 5 sec; 72°C, 45 sec.
72°C, 1 min final extension.
4°C hold
Run usually takes 1.3 hours to complete.
Remove samples, and store at 20°C until clean-up.
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PCR CLEAN-UP & QUANTITATION
1. Labeled PCR reaction is cleaned using BioLine SureClean. Resuspend SureClean pellet in 20 µL PCR-grade water. Warm to 50°C for 15 minutes to solubilize. Vortex, and store at -20°C.
2. 1 µL and 4 µL aliquots of the PCR product can be quantitated by: (1) 260nm spectrophotometry, using the
NanoSpec; (2) gel electrophoresis, along with aliquots of size and quantity standards (e.g. 5 µL of BioLine
Ladder I contains 50 ng/band).
RESTRICTION DIGESTION
3. Each TRFLP restriction digest requires 200 ng of PCR product.
4. Select a 4-cutter restriction enzyme a. Msp I - CCGG b. Dde I - GTNAG c. Hae III – GGCC d. Hha I – GCGC e. Hinf I – GANTC f. Rsa I – GTAC
5. Set up a restriction digest:
10x restriction buffer
20x BSA
PCR product DNA water restriction enzyme (5 units)
TOTAL VOLUME
____ µL
____ µL
____ µL
____ µL
____ µL
____ µL
Incubate at 37°C for at least 3 hours.
SAMPLE CLEAN-UP FOR T-RFLP GEL
6. Restriction digest is cleaned with BioLine SureClean + Colorless Co-precipitant. Do two (2) 70% Ethanol washes , using 200 µL of Ethanol each time. Air-dry in warm heatblock for 15 min or until dry.
7. Resuspend the pellet in 20 µL Beckman SLS (warming to 50°C for 15 minutes helps).
8. Mix 5µL of resuspended digest + 30 µL SLS + 0.25 µL of CEQ-600 labeled size standard.
9. Load samples into Beckman CEQ8000 sample plate, and top with one drop of mineral oil.
10. Continue with CEQ8000 run setup, using CEQ FRAG4-15sec injection run protocol.
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