Phase  

RFLP Protocol for Estimating Protistan Clone Library diversity

 Details  

This procedure is designed to screen cloned 18S rDNA genes from a library of 96 plasmid samples. Why 96? This is a convenient format in which to process a relatively large number of samples. Generally, as effort increases (more clones analyzed), diversity estimates (number of unique band patterns) also increase until one reaches the “true” community diversity. In practice, there will be some repetition of sequences within a clone library of 96. Also, there may be some “insert positive” clones, which were selected by black/white screening (S/Gal agar, Sigma), but don’t actually contain an 18S insert.  By performing RFLP analysis we obtain a diversity estimate for a particular mixed-DNA sample thereby reducing our sequencing effort by grouping clones within a particular library into RFLP families of identical banding pattern.

All enzymes are purchased from NEB. Enzymes were chosen because of their compatible reaction conditions; NEB Buffer 3, BSA and incubation at 37°C. The enzyme Not I releases the 18S rDNA insert from the plasmid but does not cut the insert sequence. Hinc II &  Sty I cut the insert into fragments for RFLP analysis. Note: If screening clones for insert of correct size (no RFLP analysis) then perform digests with only Not I and adjust the amount of water used accordingly.

Restriction Enzyme Master Mix

Prepare Master-Mix in a sterile 2 ml centrifuge tube. Set-up digests in sterile PCR plate. Add DNA to all wells then 17 µl of MM. Mix reaction with pipette tip. Digest samples overnight at 37°C.

Electrophoresis

• Prepare 4 liters of 1x TBE from 10x stock which is enough buffer for two 15-cm gels and two large BioRad gel chambers, plus enough to prepare Sybr gold gel stain.
• Prepare a 3% NuSieve (Cambrex) agarose gel for RFLP. Add 4.5 g NuSieve agarose to a clean 500 ml Erlenmeyer flask. Add 150 ml of 1x TBE. Invert a small (clean) Erlenmeyer flask over the gel containing flask and microwave on high for 2 minutes. Allow gel to cool until ~ 60°C.
• Level a gel-tray in a casting chamber, insert a 20-place thin (0.75 mm) comb. Pour the gel, allow to harden for at least 20-30 minutes. Note: A total of 5 gels will be needed for analysis of 96 clones.
• When gel has solidified release it from the casting chamber and transfer it to an electrophoresis chamber. Flood wells with 1x TBE, fill chamber until highest point of gel is submerged by 2-3 mm of buffer.
• Add 4 µl of Promega Blue/Orange loading dye (6x) to each well, mix gently on plate shaker. Load 12 µl of mixture into lanes 2-10 & 12-19. Load 10 µl of Hi-Lo Marker (Minnesota Molecular) into lanes 1, 11 & 20. Run gel for 10 minutes at 50 Volts, then 300 minutes at 100 Volts.
• Stain gels in 1X Sybr Gold (Molecular Probes) prepared in TBE buffer for approximately 30 minutes
• Photograph with VersaDoc (BioRad) system. Save image as Inverted (white background with black bands). Analyze with Diversity Database (BioRad). Gel should look something like this:

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