CTO optimization.

There is an Alu ! cut site / on one variant of the degenerate reverse primer. The fluor is on the reverse primer. Did not check the primers for restriction sites earlier. Relieved there is only one.

PCR PRODUCT AND DIGESTS:

SAMPLES: environmental samples A, F, and G from 2001 were diluted 1:10 or 1:20 and I microliter of the dilution was used as target for 16S rDNA amplification in Amplisure solution #7.

CYCLING CONDITIONS: .....

CTO TARGET: One microliter of the above 16S rDNA product (no cleanup) was used as a target for each replicate of the CTO nested amplification.

CYCLING CONDITIONS: .....

CLEANUP: For TRFLP the CTO PCR was done in triplicate, the three 50 mcl replicates were pooled, cleaned up in a Qiagen PCR cleanup kit, and eluted in 50 mcl of water.

DIGEST: 10 microliters of each of these eluates/cleaned PCR products were digested with each the following: Alu I, Hae III, Hpa II, and Sau 3A1. (will provide units of enzymes later - took care to use a modest excess (~1.5x) of that required to digest 1 mcl of DNA as described in Gibco catalog).

TRFLP REPORT LABELING CONVENTIONS

Samples are as described above but with additional sample AX, which is the direct (non-nested) CTO amplification of sample A DNA.

There are also two digests (Hae III and Hpa II) of a nested CTO run on 1 mcl of a 1:50 dilution of sample A 16S DNA..

Numbering convention on graphs (1-4 correspond to enzymes in alphabetical order. With the exception of AX1-4 only last two numbers are informative):

A to F 1 = Alu I; A to F 2 = Hae III; A to F 3 = Hpa II; A to F 4 = Sau 3A1I