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transferase activity of Taq polymerase) to a vector with single overhanging 3'
deoxythymidine (T) residues.
The following protocol is outlined in the TOPO TA Cloning Version M
instruction manual (Invitrogen): 2 pl of PCR product was added to a solution of I µ1 salt
solution, 1 µl TOPO vector and 2 pl sterile reverse osmosis water. This solution was
incubated on ice for 5 min. Following incubation, 2 pl of the TOPO cloning mix was
added to a vial of One Shot® chemically competent Escherichia coli (TOPO I OF) and
mixed gently. This was then incubated on ice for 5 min, heat shocked at 42°C for 30 sec
and then transferred back to ice and 250 pl of room temperature SOC medium
(Invitrogen) was added and the tubes were shaken (200rpm) at 37°C for 1 h. Following
incubation 50 µl was spread on pre-warmed selective agar plates (LB media (Oxoid) with
10 g L-1 agar (Oxoid) and 50µg ml-1 ampicillin (Sigma) and 40 µ1 of 40 mg ml-1 X-gal
(Sigma)) and incubated at 37°C overnight. Following incubation, white colonies were
picked and cultured overnight in selective LB medium broth (Oxoid; 50µg ml-1
ampicillin). Following incubation, the plasmid was isolated using the QIAprep® plasmid
extraction kit (QIAGEN), which uses a protocol involving the alkaline lysis of bacterial
cells (modified from Birnboim & Doly, 1979) followed by the adsorption of the DNA on
to a silica membrane in the presence of a high salt concentration. DNA is then washed by
buffers PB and PE which remove endonuclease and other contaminants. The plasmid was
digested with EcoR I to excise the PCR product, this was then analysed on an
electrophoresis gel (section 2.9.2) to determine if the cloning was successful. If cloning
was successful then the plasmid extracts were sent for sequencing (see section 2.9.6.4).
2.9.6.4 -
Sequencing reaction
The chain-termination method (Sanger et al., 1977) was used to sequence the
purified 16S rDNA PCR products, this was performed by the Nottingham University
sequencing department. The chain-termination method requires single stranded DNA.
The primers used for the amplification of the PCR product (1.5F and 1.5R - section
2.9.3.1) were used in the sequencing reaction. For sequencing of the cloned PCR product,
the M 13 forward and reverse primers were used.
The sequencing data was edited using the computer program Seqman (DNAstar
Inc. ) The data for the individual sequences and for the primed sequence areas were
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