1. Laboratory: Unit 3: purify PCR product (55-65) Lecture: DNA sequencing In-Class Writing: discuss abstracts (pages 68, 157) & AEM 63: 2647-53, 1997 Hand In: nothing Read: sample problems (pages 135-150) Due Next Class: nothing

4. Dye Primer Sequencing

6. Dye Terminator Sequencing

8. Enterococcus faecium

10. Lactobacillus paracasei

11. Sequence polymorphism in Lactobacillus paracasei 16S rRNA genes

12. mixed template

14. DNA Preparation for Sequencing DNA must be free of contaminants. Submit samples in dH 2 O or Tris, not Tris/EDTA.

15. DNA Preparation for Sequencing Remove unincorporated dNTPs & primers (Qiagen kit). 10 µL of DNA/reaction @ 50 ng/uL = 500 ng of amplicon in 10  L

16. DNA Preparation for Sequencing Estimate PCR product concentration by agarose gel electrophoresis. Compare to DNA Mass Ladder.

17. DNA Preparation for Sequencing Difficult to estimate PCR product concentration with conventional spectrophotometer. Use nanodrop spectrophotometer to estimate DNA concentration.

18. Sequencing Primers Primers (10 µL/reaction) @ 10 µM = 10 pmol/µL = 61.6 ng/  L for 8-27F primer (MW = 6161)

19. Tips for Primer Design 18-22 bases long GC content = 50-60% Annealing temperature 50-65 o C. Avoid 3 identical contiguous bases. Sequencing tandem repeats difficult. Primers should not anneal near repeats.

20. Purify PCR product prior to sequencing binding buffer (PB) = high salt wash buffer (PE) = high salt + ethanol

21. Purify PCR product prior to sequencing 50 ul distilled water dissolve in 50 ul distilled water ready for sequencing

22. Use a yellow tip to remove drops of ethanol trapped on the rim above the filter before you elute the DNA with water.

23. Low Mass Ladder (Invitrogen)

24. ng: 200 120 80 40 20 (10) bp: 2000 1200 800 400 200 (100) 2% 3:1 agarose gel

25. Although the Amazon Basin is well known for its diversity of flora and fauna, this report represents the first description of the microbial diversity in Amazonian soils involving a culture-independent approach. Among the 100 sequences of genes coding for small-subunit rRNA obtained by PCR amplification with universal small-subunit rRNA primers, 98 were bacterial and 2 were archaeal. No duplicate sequences were found, and none of the sequences had been previously described. Eighteen percent of the bacterial sequences could not be classified in any known bacterial kingdom. Two sequences may represent a unique branch between the vast majority of bacteria and the deeply branching, predominantly thermophilic bacteria. Five sequences formed a clade that may represent a novel group within the class Proteobacteria. In addition, rRNA intergenic spacer analysis was used to show significant microbial population differences between a mature forest soil and an adjacent pasture soil.