Phase  

T-RFLP Protocol for 18S rDNA on the Beckman CEQ8000 Capillary Sequencer

 Details  

Note: Protocol is an adaptation of the CME T-RFLP protocol for capillary electrophoresis (Grüntzig et al., 2002).

• Perform PCR with Beckman D4-labeled (5’-end) forward 18S rDNA primer and unlabeled reverse primer. Custom D4-labeled primers are ordered from Proligo.com. The PCR product should be less than ~640 bp in size for accurate sizing. Run 3-5 replicate amplifications for each sample to ensure sufficient product recovery for multiple restriction enzyme digestions.
• Final concentration of PCR components in 50 μl total volume:
  • D4-Primer Fwd: 0.5 μM
  • Primer Rev: 0.5 μM
  • Promega 10X buffer B: 1X
  • Promega MgCl2: 2.5 mM
  • Promega DNTP (5 mM ea.): 250 μM
  • BSA (Sigma A-7030): 300 ng/μl (Kirchman et al., 2001)
  • Promega Taq in buffer B: 2.5 units
  • DNA template: ~10 ng
• Thermal protocol: 95°C 2 min. (1x), 95°C 30 sec: 55°C 30 sec: 72°C 1 min (35x), 72°C 5 min (1x), 4°C (hold)
• Concentrate replicate PCR samples with Mo’Bio UltraCleanÔ PCR Clean-up Kit following the manufacturer's protocol, elute sample from the column in 50 μl water or Tris (supplied with kit).
• Digest entire 50 μl of concentrated PCR product with mung bean nuclease in a total volume of 100 μl (add nuclease and sterile water) to remove partially single-stranded PCR products that may contribute to T-RFLP artifacts (Egert and Friedrich, 2003).
• Concentrate the mung bean digest with Mo’Bio UltraCleanÔ PCR Clean-up Kit following the manufacturer's protocol, elute sample from the column in 50 μl water or Tris (supplied with kit).
• Band-isolate the recovered DNA on a 1.2% agarose gel (TBE) and purify the DNA from the gel with the Mo’Bio UltraCleanÔ 15 DNA Purification Kit. Use 10 μl of UltraBindÔ silica matrix to capture DNA from the melted DNA solution. Elute DNA from silica matrix with sterile water (20 μl).
• Digest DNA (100-300 ng) with 10U of HaeIII, HhaI, MnlI or MspI (or any other type II “4-base cutter”) for recommended time at appropriate temperature. Heat inactivate digests when complete. Digests are generally 20 μl total volume.
• Purify T-RFLP digest prior to running on CEQ using either the “individual PCR tube” or “plate format” the following protocol.

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Phase  

T-RFLP Protocol for 18S rDNA on the Beckman CEQ8000 Capillary Sequencer

Details  

• Add 1 μl glycogen (20 mg/ml) and 2 μl 3M NaOAc per 20 μl digest. (a master mix may be prepared for this solution)
• Add 2.5x the volume of the digest (50 μl) of ice-cold 95% Ethanol.
• Spin for 15 minutes at 4°C and max RPM (14,000 RPM on Eppendorf)
• Decant or pipette off the supernatant being very careful not to disturb the DNA pellet (if visible).
• Add 100 μl of ice-cold 70% Ethanol.
• Spin for 5 minutes at 4°C and max RPM (14,000).
• Decant or pipette off the supernatant.
• Optional: Repeat 5-7.
• Air dry (~15 minutes) in the hood and/or 37 °C incubator
• Resuspend pellet in 40 μl Beckman SLS (formamide)

96-well plate format

• Add 1 μl glycogen (20 mg/ml) and 2 μl 3M NaOAc per 20 μl digest. (a master mix may be prepared for this solution)
• Add 2.5x the volume of the digest (50 μl) of ice-cold 95% Ethanol.
• Spin for 20 minutes at 4°C and max RPM (5,700)
• Perform 20 sec. inverted spin at 300 RPM
• Add 100 μl of ice-cold 70% Ethanol.
• Spin for 10 minutes at 4°C and max RPM (5,700).
• Perform 20 sec. inverted spin at 300 RPM
• Optional: Repeat 15-17.
• Air dry (~15 minutes) in the hood and/or 37 °C incubator
• Resuspend pellet in 40 μl Beckman SLS (formamide)
• Dilute sample (e.g, 5 μl resuspended product plus 35 μl SLS) prior to running on the CEQ, actual dilutions may vary from the example (see Table below).

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Phase  

Examples of dilutions of resuspended digest to load onto CEQ

Details  

Note: SLS (sample loading solution) is Beckman deionized formamide.

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Phase  

CEQ8000 Run Protocols

Details  

CEQ run Method: Modified Frag-4
Injection time: Variable (generally 10-20 sec.)
Separate:

• Stage 1: Ramp to 2kV over 2 minutes
• Stage 2: Switch to 4.9kV at 2 minutes and run at 4.9kV and 60°C for 60 minutes.

References:
Egert, M., and M. W. Friedrich. (2003) Formation of pseudo-terminal restriction fragments, a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure. Applied and Environmental Microbiology 69:2555-2562.
Grüntzig, V., B. Stres, H. L. Ayala del Río, and J. M. Tiedje (2002) Improved protocol for T-RFLP by capillary electrophoresis. Center for Microbial Ecology, Michigan State University East Lansing, Michigan, 48824 (http://rdp.cme.msu.edu)
Kirchman, D. L., L. Y. Yu, B. M. Fuchs, and R. Amann. (2001) Structure of bacterial communities in aquatic systems as revealed by filter PCR. Aquatic Microbial Ecology 26:13-22.

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