Table 1

Comparison of LSplex labelling methods

Labelling Method

Description

Final amount of DNA1 (μg)

Base/Dye ratio2

Labelled nucleotides

Processing time


Random Priming

labelling after amplification with Klenow DNA polymerase

1.3

61

dCTP-Cy3

1.5 h LSplex, 15 min purification; 2 h labelling, 15 min purification

Chromatide

direct incorporation of fluorescent nucleotides during Lsplex

0.7

139

Alexa Fluor 546-14-dUTP(1:3)3

1.5 h LSplex, 15 min purification

ARES

incorporation of amino-modified nucleotides during Lsplex staining with Amino-reactive dye

1.1

64

aminoallyl-dUTP (1:2)4 stained after PCR with Alexa Fluor 555

1.5 h Lsplex, 15 min purification; 1 h post staining, 15 min purification


1. Amplified DNA estimated after the last purification step. The starting material for all protocols was 10 ng genomic S. aureus DNA (ATCC 29213)

2. BDR calculated following the formula: base:dye = (Abase × Єdye)/(Adye × Єbase); Abase = A260 - (Adye × CF260) Єdye is the extinction coefficient for the fluorescent dye (Cy3: 150000 cm-1M-1; Alexa555: 150000 cm-1M-1; Alexa 546: 104000 cm-1M-1) Єbase here is the average extinction coefficient for a base in double strand DNA (6600 cm-1M-1) CF: Correction Factor Cy3: 0.08; Alexa 555: 0.04; Alexa 546: 0.21

3. Ratio recommended by the manufacturer for PCR labelling

4. The manufacturer does not provide a protocol for PCR labelling

Palka-Santini et al. BMC Microbiology 2009 9:1   doi:10.1186/1471-2180-9-1

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