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| Hello all, I'm looking to clone and sequence 16s rDNA TRFLP bands. What a nightmare! I'm running IR-labelled PCR product digests on a Li-Cor acrylamide based DNA sequencer. With the help of an IR viewer I can cut out the DNA fragments from the gel. The problem is in the cloning and sequencing of the bands. I must go through a cloning step first for many of the bands, since I may have 2 or more distinct PCR digestion products co-migrating at the same bp resolution. In addition the samples have been denatured, and are at very low quantities, and for reamplification I only know the sequence for one priming site. Some procedural thoughts: 1. Cut band out of gel, re-amplify/second strand synthesis with classical random priming or phi29 based random amplification (any thoughts?), use previously engineered restriction site within primer to cut off the 5' IR-label (the other end is already blunt), then clone into dephosphorylated vector. 2. Cut bands out of gel, re-anneal (with the hope that similar sequences don't form chimeras), digest then clone? I'm guessing low nanogram to sub-nanogram quantities here. 3. Cut bands out of gel, re-anneal (with the hope that similar sequences don't form chimeras), ligate in an adaptor to the end without a known priming site, re-pcr, and clone? 4. Quit science, concentrate more on mountain biking here in Colorado, let my wife deal with making money. Gary |
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| 16s , bands , cloning , rdna , trflp |
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