BIO 535
Wong
T-RFLP Analysis
PCR clean-up & quantitation
1. Labeled PCR reaction is cleaned using SureClean Plus (BioLine).
 add 3 µL of pink coprecipitant to DNA sample
 add volume of SureClean equal to that of DNA sample
 incubate at room temp for 10 min.
 centrifuge at maximum speed (~13000 rpm) for 15 min.
 carefully pipet off supernatant without disturbing pellet
 add at least 100 µL cold 70% Ethanol
 centrifuge at maximum speed (~13000 rpm) for 15 min.
 carefully pipet off supernatant without disturbing pink pellet
 vacuum-dry pellet for 15 min
 resuspend in desired volume of PCR-clean water
2. 1 µL and 4 µL aliquots of the cleaned DNA are quantitated by gel electrophoresis, along
with aliquots of size and quantity standards (e.g. BioLine Ladder I is 10 ng/µL).
Restriction digestion
3. Digest 100 ng of PCR product for TRFLP.
4. Select a 4-cutter restriction enzyme
 Msp I - CCGG
 Dde I - GTNAG
 Hae III – GGCC
 Hha I – GCGC
 Hinf I – GANTC
 Rsa I – GTAC
5. Set up a restriction digest:
10x restriction buffer
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20x BSA for restriction digests
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PCR product DNA
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water
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restriction enzyme (5 units)
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Total volume
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Incubate at 37°C (or recommended temp) for at least 3 hours.
Sample preparation for TRFLP gel
6. Clean the digest using SureClean Plus as before. Pink pellet should be evident.
7. Resuspend the pellet in 20 µL SLS (Beckman-Coulter). If pellet doesn't readily dissolve,
warm sample in heat block and vortex repeatedly.
8. For each well in a CEQ sample tray, add 10 µL digested sample + 0.25 µL CEQ 400 or
CEQ 600 bp size marker + 30 µL SLS (best way is to make a master mix of SLS + size
marker, and aliquot for each sample)
9. Remove air bubbles in sample well, and overlay with mineral oil.
10. Consult CEQ run protocol for capillary electrophoresis. Use a FRAG-4+15sec injection
profile to avoid overloading capillaries.