This is an idea for a protocol for performing site-directed mutagenesis. The first step is generation of the ssDNA template. The second step is the mutagenesis.
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Transform pET29b(+) harboring your construct into Escherichia coli CJ236
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Plate on chloramphenicol (25 ng/µL) and kanamycin (50 ug/µL) selection plates
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Grow 2 x 250 mL cultures (you will get about 1 µg plasmid for each 1 mL of culture so even 2 x 100 mL is plenty)
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Miniprep plasmids. For each miniprep, use 5-10 mL culture. You can load multiple samples onto one column and get very concentrated DNA, or spread out, or midi- or maxiprep instead.
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Dilute the plasmid solution to 60 ng/µL
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Dilute the ExoIII 1/10 in CutSmart buffer
- minirecipe
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Set up template preparation reaction. Per 50 µL,
- 40.8 µL plasmid DNA (at 60 ng/µL)
- 5 µL 10X CutSmart buffer
- 2 µL Nb.BsrDI (10 units/µL)
- 2 µL Exonuclease I (20 units/µL)
- 0.2 µL Exonuclease III (100 units/µL)
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Set up as many reactions as desired
- Load all the reactions into 2 miniprep columns
- (Do additional wash step? Can't see how it helps)
- Wash with ethanol buffer
- Spin again to remove ethanol
- Elute into tubes
- Determine concentration using A280
Note: the theoretical maximum yield of this procedure is 1250 ng of DNA, so resuspend in 50 µL or so so get a good working concentration (25 ng/µL) for Kunkel reactions (each Kunkel will require 1 µL).
- Pour a 0.8% agarose gel (800 mg agarose + 100 mL TAE + 10 µL SYBR Green II)
- Run both samples and diluted intact plasmid
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Based on ideas from Emily Wrenbeck (MSU) and Wai Shun Mak (UCD)