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April 19, 2013
Quikchange Site Directed Mutagenesis
- The QuikChange Site-Directed Mutagenesis Kit (Catalog #200519) contains enough reagents for 10 total reactions, which includes 5 control reactions.
- Thaw the dNTP mix once, prepare single-use aliquots, and store the aliquots at –20°C. Do not subject the dNTP mix
to multiple freeze-thaw cycles.
- Use 125 ng of each primer
- 10× Reaction Buffer
- 100 mM KCl
- 100 mM(NH4)2SO4
- 200 mM Tris-HCl (pH 8.8)
- 20 mM MgSO4
- 1% Triton® X-100
- 1 mg/ml nuclease-free bovine serum albumin (BSA)
- TE Buffer
- 10 mM Tris-HCl (pH 7.5)
- 1 mM EDTA
- Control Reaction
- 5 μl of 10x reaction buffer
- 2 μl (10 ng) of pWhitescript 4.5-kb control template (5 ng/μl)
- 1.25 μl (125 ng) of oligonucleotide control primer #1 [34-mer (100 ng/μl)]
- 1.25 μl (125 ng) of oligonucleotide control primer #2 [34-mer (100 ng/μl)]
- 1 μl of dNTP mix
- ddH2O to a final volume of 50 μl
- Sample Reaction
- 5 μl of 10× reaction buffer
- X μl (5–50 ng) of dsDNA template
- X μl (125 ng) of oligonucleotide primer #1
- X μl (125 ng) of oligonucleotide primer #2
- 1 μl of dNTP mix
- ddH2O to a final volume of 50 μl
- Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to each control and sample reaction
- Thermal cycling: 95°C, 30 sec; [95°C, 30 sec; 55°C, 1 min; 68°C, 7 min (1 min/kb plasmid length)]x18
- Add 1 μl of the Dpn I restriction enzyme (10 U/μl)
- Gently and thoroughly mix each reaction, spin down in a microcentrifuge for 1 minute, and immediately incubate at 37°C for 1 hour to digest the parental supercoiled dsDNA
- Transform 1 μl of the Dpn I-treated DNA from each control and sample reaction into separate 50-μl aliquots of XL1-Blue supercompetent cells
- As an optional control, verify the transformation efficiency of the XL1-Blue supercompetent cells by adding 1 μl of the pUC18 control plasmid (0.1 ng/μl) to a 50-μl aliquot of the supercompetent cells
- Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30 minutes
- Heat pulse the transformation reactions for 45 seconds at 42°C and then place the reactions on ice for 2 minutes
- Add 0.5 ml of NZY+ broth preheated to 42°C and incubate the transformation reactions at 37°C for 1 hour with shaking at 225–250 rpm
- Plate entire 250 μL volume and incubate for at least 16 hours
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