Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.
Fosmid library construction has been divided into four main steps and sub-divided into several parts (see Fig.1 for an overview).
Step I (see protocol “DNA extraction from 0.22 μM Sterivex filters” [3])
Part 1: Enzyme-catalyzed cell lysis
Part 2: Purification of environmental DNA by CsCl density gradient centrifugation and DNA recovery
Part 3: Quality control of extracted DNA by gel electrophoresis
Step II
Part 1: Enzymatic modification steps of recovered environmental DNA
All reagents necessary for the end-repair step of the environmental DNA are included in the pCC1 Fosmid Library Production Kit from EPICENTRE. Ideally, your genomic DNA should be adjusted to 0.5 μg/μl.
Part 2: Size-selection of genomic DNA by pulsed-field gel electrophoresis (PFGE)
Part 3: DNA-recovery by gel extraction and ligation of recovered DNA to the fosmid cloning vector
All reagents for this part are included in the EPICENTRE fosmid-library production kit.
Step III
Part 1: Phage-packaging of vector-insert ligation product
Part 2: Infection of the library host E. coli
Part 3: Plating and titering
Step IV
Part 1: Agar- and well plates preparation
Part 2: colony picking robot setup
Part 3: Overnight incubation and library storage
Representative Results:
The presented protocol describes the procedure how to generate an environmental fosmid library to capture the genetic content of a microbial community in a given habitat. This protocol should create a fosmid library which is representative to the genetic content of the sampled environment and should enable the reader to modify and optimize critical steps if the amount of fosmid clones obtained at the end of the whole procedure is too low.
A procedure is described how to most efficiently generate a large-insert fosmid library with genomic DNA derived from a coastal water sample. Up-stream genomic DNA extraction is described in a separate protocol [3].
As the fosmid-library production is a multistep-process, plan at least two to three weeks in time for the whole procedure including all four presented steps. The extraction of genomic DNA is the most crucial step and all down-stream steps rely on the quality and quantity of extracted genomic DNA; so consider spending enough time for this step and work carefully. Quality and quantity control of your extracted DNA is very important. It can happen that the number of fosmid clones is low especially if this is the first library you are going to make. As the fosmid library construction by itself is straight forward, failure is mainly caused by insufficient quality and/or quantity of your extracted and purified genomic DNA. Consider to re-extract genomic DNA and pay special attention to the steps involved in purification and DNA-recovery if your library construction was not successful.
The authors have nothing to disclose.
We would like to thank the Canadian Foundation for Innovation, the British Columbia Knowledge Development Fund and the National Sciences and Engineering Research Council (NSERC) of Canada for supporting ongoing studies on low oxygen regions of coastal and open ocean waters. M.T. and S.L were supported by fellowships from the TULA foundation funded Centre for Microbial Diversity and Evolution (CMDE). M.T. also received fellowship support from the Deutsche Forschungsgemeinschaft (DFG).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Step II | ||||
CopyControl™ Fosmid Library Production Kit | EPICENTRE | CCFOS110 | sufficient to generate 9-10 fosmid libraries | |
SeaPlaque Agarose | Cambrex | 50100 | ||
stirring hot plate | Corning | PC-420D | ||
1.0X TAE running buffer | ||||
CHEF Mapper XA pulsed field electrophoresis system including cooling module and variable speed pump | Bio-Rad | |||
λ DNA-HindIII Digest | NEB | N3012S | ||
MidRange I PFG Marker | NEB | N3551S | ||
10,000X SYBR Gold | Invitrogen | S11494 | ||
microwavable plastic wrap | Saran premium wrap | |||
Safe Imager transilluminator | Invitrogen | S37102 | ||
GELase Agarose Gel-Digesting Preparation | EPICENTRE | G09100 | ||
scalpel | Fisher Scientific | 08-927-5D | ||
Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane | Millipore | UFC801024 | ||
Microcon YM-30 Centrifugal Filter Unit | Millipore | 42410 | ||
nuclease free water | Ambion | AM9932 | ||
Quant-iT PicoGreen dsDNA Assay Kit *2000 assays*# | Invitrogen | P7589 | ||
digital dry block heater | VWR | 12621-088 | ||
water bath | VWR | 14231-854 | ||
centrifuge | Beckman Coulter | Avanti J-E | ||
centrifuge rotor | Beckman Coulter | JA 5.3 | ||
table top centrifuge | Eppendorf | 5415D | ||
microcentrifuge tubes, 1.7 mL, clear | Axygen | MCT-175-C | ||
15 mL centrifuge tubes | Fisher-Corning | 430052 | ||
Step III | ||||
LB broth | Fisher Scientific | BP 1426-2 | ||
MgSO4 | Sigma-Aldrich | M2643 | ||
phage dilution buffer | 10 mM Tris-HCl [pH 8.3], 100 mM NaCl, 10 mM MgCl2 | |||
cryogenic vials | Fisher-Corning | 430488 | ||
Step IV | ||||
96 well plate with lid | Fisher-Corning | CS003370(3370) | ||
384 well plate with lid | Fisher-Corning | 07201157(3680) | ||
Petri dishes 100 O.D. x 15mm H | Fisher | 08-757-12 | ||
Petri dishes 150 Dia. x 15mmH | Fisher | 08-757-14 | ||
BD Falcon BioDish XL 245 X 245 mm | VWR | CABD351040 | ||
glycerol | Sigma-Aldrich | G5516 | ||
chloramphenicol | Sigma-Aldrich | C0378 | ||
LB broth | Fisher Scientific | BP 1426-2 | ||
Difco Agar | BD | 214530 | ||
glass beads | Fisher Scientific | 10-310-1 | ||
QFill3 | Genetix | X3050 | ||
Bleach | Javex | |||
Ethanol | Sigma-Aldrich | E7023 | ||
15 mL centrifuge tubes | Fisher-Corning | 430052 | ||
QPix colony picker | Genetix | QPix2 | ||
picking head (E. coli) | Genetix | X4006 |