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.

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Presentation transcript:

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

Dye Primer Sequencing

Dye Terminator Sequencing

Enterococcus faecium

Lactobacillus paracasei

Sequence polymorphism in Lactobacillus paracasei 16S rRNA genes

mixed template

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

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

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

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

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

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

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

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

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

Low Mass Ladder (Invitrogen)

ng: (10) bp: (100) 2% 3:1 agarose gel

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.