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Transposon Tools and Linear Target DNA
Why linear target?
The Finnzymes transposon tools insert a copy of the artificial transposon, Entranceposon, efficiently into linear dsDNA (e.g. restriction fragment, PCR product) as well as in circular DNA. This feature may come handy in some specific applications, for example in the sequencing of small cDNA inserts in which the unwanted transposon insertions in the cloning vector backbone occur at a relatively high frequency.
The protocol steps in short:
1. Production of the linear target
2. In vitro transposition reaction
1. Production of the linear target
Amplify your linear target by PCR using primers that contain recognition sites for restriction enzymes that yield sticky ends upon cleavage. Alternatively digest your plasmid with restriction enzymes to release the linear target fragment.
Important: Avoid using restriction enzymes that produce 5'-GATC-3' sticky end (BamHI, BglII). This is vitally important because the unreacted Entranceposon fragment present in the transposition reaction mixture ligates efficiently into 5'-GATC-3' sticky ended and blunt ended cloning vector. This will most likely result in a large number of false positives in the subsequent transformation step (see figure 2 a.). If your target DNA as well as your vector are blunt-ended it may be necessary to treat the Entranceposon with alkaline phosphatase (CIP) before the transposition reaction in order to prevent the false ligation.
Extract the target fragment from agarose gel to get rid of the vector fragment or the PCR template plasmid.
2. In vitro transposition reaction
Perform the transposition reaction according to the Instruction Manual of TGS™ II Kit (pdf), MGS™ Kit (pdf) or STOP™ Kit (pdf).
The in vitro transposition complex is assembled as MuA Transposase is added to the reaction with the artificial transposon, Entranceposon (a.). The MuA Transposase enzyme catalyzes all the chemical reactions that are required for inserting a copy of the Entranceposon into target DNA (b.). As a result the Entranceposon is covalently joined to the linear target DNA at a random site (c.).
Important: The Entranceposons in the TGS II Kit (F-702), MGS Kit (F-701) and STOP Kit (F-703) kits are compatible with linear target DNA. However, we recommend not to use the Entranceposons included in the original TGS I Kit (Kit F-700, Entranceposons F-751 CamR or F-759 KanR) with linear target DNA. These two Entranceposons were specifically produced for sequencing circular targets.
We have found that it is useful to scale up the transposition reaction to yield enough reaction products for the ligation step (for example up to 5x).
Desalt/precipitate the transposition reaction mixture prior to ligation reaction (Thomas, M. R. (1994). Simple, effective cleanup of DNA ligation reactions prior to electro-transformation of E. coli. BioTechniques 16, 988.):
Add dH2O to 50 µl (1x transposition reaction, 20 µl)
Add 500 µl (10 volumes) n-butanol
Vortex for 10-20 s
Centrifuge at 12-14 000 rpm for 15 min
Discard supernatant, dry pellet
Resuspend pellet in 10 µl dH2O
Ligate the desalted transposition reaction mixture into a digested vector. Use 1-5 ng of the vector per 1x transposition reaction mixture. We have successfully used 10 ng of pUC19 as a cloning vector with the desalted reaction products from a 5x transposition reaction.
In most cases it is useful to desalt/precipitate the ligation mixture prior to electroporation as described above for the transposition reaction mixture.
Heat inactivate the ligase 10 min at 65 ºC
Add dH2O to 50 µl (ligation reaction volume 10-20 µl)
Add 500 µl (10 volumes) n-butanol
Vortex for 10-20 s
Centrifuge at 12-14 000 rpm for 15 min
Discard supernatant, dry pellet
Resuspend pellet in 10 µl dH2O
Use 2-10 µl of the desalted ligation mixture per electroporation
Use electrocompetent cells with a high transformation efficiency (>108 cfu/µg pUC19) in order to ensure recovery of an adequate number of insertion clones. Follow the specific instructions of your electroporation apparatus. Immediately after the electroporation pulse add SOC medium ad 1 ml and incubate for 1 hour at 37 ºC. Spread aliquots on LB plates supplemented with the antibiotics that select for the Entranceposon and your vector.
