Invitrogen Bac to Bac Expression System

Analyzing Recombinant Bacmid DNA by PCR
Introduction
Recombinant bacmid DNA is greater than 135 kb in size. Since restriction analysis
is difficult to perform with DNA of this size, we recommend using PCR analysis
to verify the presence of your gene of interest in the recombinant bacmid. The
bacmid contains M13 Forward (-40) and M13 Reverse priming sites flanking the
mini-attTn7 site within the lacZα-complementation region to facilitate PCR
analysis (see figure below). Guidelines and instructions are provided in this
section to perform PCR using the M13 Forward (-40) and M13 Reverse primers.
Transposed pFastBac
sequence
Tn7R Gene of interest Tn7L
Bacmid DNA
M13 (-40)
Forward
128 bp 145 bp
mini-attTn7
M13
Reverse
PCR Analysis with
M13 Primers
To verify the presence of your gene of interest in the recombinant bacmid using
PCR, you may:
• Use the M13 Forward (-40) and M13 Reverse primers
• Use a combination of the M13 Forward (-40) or M13 Reverse primers and a
primer that hybridizes within your insert.
The M13 Forward (-40) and M13 Reverse primers are available from Invitrogen
(see below).
Primer Sequence Catalog no.
M13 Forward (-40) 5′d[GTTTTCCCAGTCACGAC]3′ N540-02
M13 Reverse 5′d[CAGGAAACAGCTATGAC]3′ N530-02
DNA Polymerase You may use any DNA polymerase of your choice for PCR including Platinum ®
Taq DNA Polymerase (Invitrogen, Catalog no. 10966-018). If the expected PCR
product is > 4 kb, we recommend using a polymerase mixture such as Platinum ®
Taq DNA Polymerase High Fidelity (Invitrogen, Catalog no. 11304-011) for best
results.
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