This protocol is desgined for use with the stratagene PfuUltra II Fusion DNA polymerase and is based in part on the PfuUltra II Fusion DNA polymerase usage manual. PfuUltra II Fusion Manual
To produce clones of two genes from B.subtilis that are too big to have synthesised by GeneArt; for use as an integration site and gene knockout (EpsE) or for their original purpose as a transcriptional regulator (XylR).
To produce clones of sequences from vectors; for use as integration sites (AmyE), antibiotic resistance (Spectinomycin) and as a transcriptional repressor (LacI).
A modified protocol for using Taq polymerase can be used if less fidelity is required
Heated lid PCR machine
Thin walled PCR tube
If a vector is used as the template, 5ng of plasmid DNA should be used instead and the volume of H2O to be added should be adjusted accordingly.
If PCR is proving difficult, particularly for denaturation, DMSO can be added to a final concentration of 10% to increase efficiency
The forward and reverse primers should contain the Biobrick prefix (forward primer) and the complementary sequence to the Biobrick suffix (reverse primer) 5' of the beginning of the annealing sequence.
Add all the reagents in order (down the list) sequentially to the PCR tube, mxing after each addition.
Place the PCR tubes into the PCR machine and set the programme to the following set-up:
The resulting solution can then be purified using a PCR purification column, by gel electrophoresis followed by spin purification or can simply be ligated ready for use.