Site-Directed Mutagenesis

Authors

Lili Jing

Summary

This protocol is for making nucleotide changes at specific loci in a large vector (>= 10 kb), and based on QuikChange II XL Site-Directed Mutagenesis Kit (stratagene).

Materials

1. QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene)
2. LB agar (Sigma)
3. Antibiotics (Sigma)

Equipment

1. Thermal cyclers (Bio-Rad)

Procedure

A. Mutant Strand Synthesis Reaction

a) Prepare the sample reaction as follows:

5 μl of 10x reaction buffer

X μl (10 ng) of dsDNA template

1.25 μl (125 ng) of oligonucleotide primer #1

1.25 μl (125 ng) of oligonucleotide primer #2

1 μl of dNTP mix

3 μl of QuikSolution

ddH2O to a final volume of 50 μl

Then add 1 μl of PfuUltra HF DNA polymerase (2.5 U/μl)

b) Cycle each reaction using the following parameters (be sure to adhere to the step 2-4 18 cycle limit)

1. 95 °C 1min
2. 95 °C 50sec
3. 60 °C 50sec
4. 68 °C 1 min/kb of plasmid length*
5. 68 °C 7 min

Note:*for example, a 5 kb plasmid requires 5 min at 68 °C per cycle

c) Following cycling, place reaction tubes on ice for 2 min to cool the reactions to 37 °C

d) Check product by electrophoresis of 10 μl of product on 1% agarose gel. While gel is running, start Dpn1 Digestion

B. Dpn1 Digestion of the Amplification Products

a) Add 1 μl of the Dpn1 restriction enzyme (10 U/μl) directly to each amplification reaction using a small, pointed pipet tip

b) Gently mix each reaction mixture by pipetting up and down several times. Spin for 1 min and incubate the reaction at 37 °C for 1 h to digest the parental supercoiled dsDNA

C. Transformation of XL10-Gold Ultracompetent Cells

a) Gently thaw XL10-Gold ultracompetent cells on ice. For each control and sample reaction to be transformed, aliquot 45 μl of the cells to a prechilled polypropylene tube

b) Add 2 μl of the B-ME mix provided with the kit to the 45 μl cells

c) Swirl the contents of the tube gently. Incubate on ice for 10 min, swirling gently every 2 min

d) Transfer 2 μl of the Dpn1-treated DNA from each control and sample reaction to separate aliquots of the ultracompetent cells
For a positive control, add 1 μl of 0.01 ng/μl pUC18 control plasmid

e) Preheat NZY + broth in a 42 °C water bath

f) Heat-pulse tubes in a 42 °C water bath for 30 sec

g) Incubate the tubes on ice for 2 min

h) Add 0.5 ml of preheated (42 °C) NZY + broth to each tube, then incubate the tubes at 37 °C for 1 h with shaking 225-250 rpm

i) Plate the appropriate volume of each transformation rxn as indicated on the table below on proper selection plates:

1. 250 μl                                  pWhitescript mutagenesis control
2. 5 μl (in 200 μl NZY + broth)  pUC18 transformation control
3. 250 μl on each of two plates (entire transformation reaction) Sample mutagensis

j) Incubate plates at 37 °C for > 16 h

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