1. 6 litres of seawater were sampled in a Nalgen® bo5le 2. 1 Litre of water was then pumped with a peristal:c pump 3. A prefiltra:on was done through 10 and 3 µm PC membrane filters 4. Prokaryotes were recolted in a 0.22 µm Sterivex® filter. COMPARISON OF THREE EXTRACTION PROTOCOLS FOR GENOMIC STUDIES OF PELAGIC BACTERIA Clarisse Lemonnier M2 Microbiologie Fondamentale et Appliquée, UBO [email protected] INTRODUCTION Cultureindependant technics are essan:al to assess marine bacterial diversity The first step is cri:cal : DNA extracAon MATERIALS & METHODES OBJECTIVES Enough DNA quality and quan:ty? Is all the diversity extracted? 1 Filtra:ons Comparison of 3 DNA extracAon applied to marine bacteria (collected on a 0,22µm Sterivex filter) 2 Extrac:ons 3 Analyses Protéinase K – 55°C Phenol chloroform MOBio PowerSoil kit S2 S3 S1 DNA quan:ty and quality Nanodrop 1000 Picogreen QuantiTTM PicoGreen® kit. Diversity analysis RESULTS & DISCUSSION ARISA kit Agilent 7500 DNA Quan:ty and Quality Diversity S1 S3 S2 PCR of ITS S1 S2 S3 S1 allows to extract the greatest DNA quanAty S3 really countains DNA DNA quan<ty extracted with S3 is not detactable by the Picogreen S3 shows the highest specific richness a b c a b c a b c + A too longer Ame of bead beaAng might explain the low DNA extracted with the S3 method. However it seems to be the most efficient technic to beWer asses diversity 510min CONCLUSION Finding the opAmal extracAon protocol is quite complex and need a lot of few rearrangements. It would be interesAng to repeat S3 and compare different Ame of beadbeaAng. 10 µm 3 µm

Lemonnier_Poster_prestage... · M2"Microbiologie"Fondamentale"etAppliquée,"UBO" [email protected]""

Download PDF Report

Upload
dangdien
View
215
Download
0

Embed Size (px)

Citation preview

Page 1: COMPARISON#OFTHREE#EXTRACTION#PROTOCOLS#FOR …pagesperso.univ-brest.fr/~maignien/doc/Lemonnier_Poster_prestage... · M2"Microbiologie"Fondamentale"etAppliquée,"UBO" lemonnier.clarisse@gmail.com""

1. 6 litres of seawater were sampled in a Nalgen® bo5le

2. 1 Litre of water was then pumped with a peristal:c pump

3. A prefiltra:on was done through 10 and 3 µm PC membrane filters

4. Prokaryotes were recolted in a 0.22 µm Sterivex® filter.

COMPARISON OF THREE EXTRACTION PROTOCOLS FOR GENOMIC STUDIES OF PELAGIC BACTERIA

Clarisse Lemonnier M2 Microbiologie Fondamentale et Appliquée, UBO

[email protected]

INTRODUCTION Ø  Culture-‐independant technics are essan:al

to assess marine bacterial diversity

Ø  The first step is cri:cal : DNA extracAon

MATERIALS & METHODES

OBJECTIVES

à  Enough DNA quality and quan:ty? à  Is all the diversity extracted?

1 Filtra:ons

Ø  Comparison of 3 DNA extracAon applied to marine bacteria (collected on a 0,22µm Sterivex filter)

2 Extrac:ons 3 Analyses Protéinase K – 55°C

Phenol-‐chloroform

MO-‐Bio PowerSoil kit

DNA quan:ty and quality

Nanodrop1000

Picogreen Quant-‐iTTM PicoGreen® kit.

Diversity analysis

RESULTS & DISCUSSION

ARISA kit Agilent 7500

DNA Quan:ty and Quality Diversity

S1 S3 S2

PCR of ITS

S1 S2 S3

S1 allows to extract the greatest DNA quanAty

S3 really countains DNA DNA quan<ty extracted with S3 is not detactable by the Picogreen

S3 shows the highest specific richness

a b c -‐ a b c -‐ a b c -‐ + -‐

A too longer Ame of bead beaAng might explain the low DNA extracted with the S3 method. However it seems to be the most efficient technic to beWer asses diversity

5-‐10min

CONCLUSION Finding the opAmal extracAon protocol is quite complex and need a lot of few rearrangements. It would be interesAng to repeat S3 and compare different Ame of bead-‐beaAng.

10 µm 3 µm