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

Dye Primer Sequencing

Dye Terminator Sequencing

Enterococcus faecium

Enterococcus faecium

Lactobacillus paracasei

Sequence polymorphism in Lactobacillus paracasei 16S rRNA genes

mixed template

mixed template

DNA Preparation for Sequencing

DNA must be free of contaminants.

Submit samples in dH2O or Tris, not Tris/EDTA.

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

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 µL/reaction) @ 10 µM = 10 pmol/µL

= 61.6 ng/L for 8-27F primer (MW = 6161)

Tips for Primer Design

18-22 bases long GC content = 50-60%

Annealing temperature 50-65oC.

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 DNAwith water.

Low Mass Ladder(Invitrogen)

ng:200

120

80

40

20

(10)

bp:2000

1200

800

400

200

(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.