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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
|10Χ PfuUltra™II reaction buffer||2.5μL|
|dNTP mix (10mM)||0.5μL|
|B.subtilis genomic DNA (100ng/μL)||1μL|
|Forward Primer (100ng/μL)||1μL|
|Reverse Primer (100ng/μL)||1μL|
|PfuUltra® II fusion HS DNA polymerase||0.25μL|
|Total Reaction Volume||25μL|
Note: Template DNA should be diluted to 100ng/μL. If template DNA concentration is below 100ng/μL, 100ng of DNA should be added and the volume of H2O to be added should be adjusted to maintain a reaction volume of 25μL.
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:
|Initial Denaturation:||30 seconds at 95°C (longer for genomic DNA)|
|10 Cycles of:||30 second denaturation at 95°C|
|30 second annealing time at Primer Tm - ~3°C (complementary section of primer Tm)|
|15 seconds (+ 15 second for each additional kb) extending time at 68C (30 seconds for genomic templates)|
|20 - 30 Cycles of:||30 second denaturation at 95°C|
|30 second annealing time at Primer Tm - ~3°C (total primer Tm)|
|15 seconds (+ 15 second for each additional kb)extending time at 68°C (30 seconds for genomic templates)|
|Final Extension:||5 minutes at 68°C|
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.