Supplementary Methods to LaMontagne and Holden 2003.

 

Substitution of PEG-8000 for ethanol in the precipitation step reduced humic acid contamination.  PEG precipitated samples had UV spectra consistent with DNA but ethanol precipitated samples did not have the characteristic trough at 230 nm, as evidenced by low A260/A230 ratios (Table A). Recovery (94%) and purity (A260/A230 = 1.51) of DNA collected by PEG precipitation was higher than ethanol precipitated samples (Table A).  RNAase treatment may have helped purify ethanol-precipitated samples by degrading humic acid/RNA complexes but this DNA was not consistently PCR-amplifiable without further purification and RNAse treatment was not separated from phenol/chloroform extraction. 

This difference in amplifiability confirms the increase in purity obtained with PEG-precipitation, which, coupled with increased yield and less variability (Table A), implies PEG precipitation is superior to ethanol precipitation. 

 

Table A. Yield and purity of DNA purified from aquatic samples collected by filtrationa and concentrated by either ethanol or PEG precipitation.

 

DNA

 

Recoveryc prior to RNAse A treatment

A260/A230 prior to RNAse A treatment

A260/A230 after RNAse A treatmentd

Site

(mg/L)b

Ethanol

PEGe

Ethanol

PEGe

Ethanol

PEGe

Creek

18.5

73%

93%

0.71

1.37

0.99

1.16

Lagoon

14.9

86%

95%

0.20

1.55

1.45

1.41

Ocean

9.0

64%

94%

0.16

1.61

1.27

1.25

 

Mean

74%

94%

0.36

1.51

1.24

1.27

 

SD

11%

1%

0.30

0.12

0.23

0.13

 

P

0.037

0.004

0.823

a.       Samples were collected on December 20th, 2000. 

b.      Measured in lysate with PicogreenÔ (Molecular Probes, Eugene, OR)

c.       Percent recovery after precipitation of lysate.

d.      Nucleic acids treated with RNAse A, phenol/chloroform extracted and ethanol precipitated by standard methods [Sambrook, 1989 #92].

e.       See PEG protocol below.

 

 

Prepare a 40% PEG 8000 (Sigma P-4463) by mixing

 40g PEG 8000 in 80 ml DI

 bring to 100 ml

 autoclave

 

1) for precipitation add PEG 8000 to concentration final of 11%.

Make sure you draw the PEG into the pipet slowly, it is viscous.

Salt (NaCl) concentration final should be 0.8 to 1.1 M.
Final PEG concentrations from 8 to 11 % work well, lower concentrations may give higher MW and relatively pure DNA, experiment with a range.

Protocol will work well with other salts for example concentration final 0.4 M K acetate

2) incubate ON at 4 C (60 min should be plenty though)

3) pellet at 12,000 G at 4 C for 15 min

4) carefully aspirate away supernatant with a pipet

5) add 0.6 volumes 70% ethanol, place tube in same orientation as first spin and spin again 12,000 G 4 C 10 min

6) carefully aspirate away supernatant