Vietnam National University-HCMC International University

Site-directed mutagenesis protocol
Lecturer : Dr. Tran Ngoc Duc

L/O/G/O

Group¶s members:
Tran Thi Anh Thuy Tran Thi Dieu Thanh Than Thi My Linh BTIU08114 BTIU08110 BTIU08127

Introduction .

usually a circular molecule known as a plasmid. followed by PCR. . Simply done by designing a ³mutated´ primer.What is Site-directed mutagenesis? A molecular biology technique in which a mutation is created at a defined site in a DNA molecule.

General procedure .

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‡ ‡ Cut up the template DNA with DpnI.General procedure ‡ Purify template plasmid DNA from a dam+ Escherichia coli strain (to ensure that all GATC sites are methylated for later digestion with DpnI). . This results in nicked circular strands of the plasmid. ‡ Run a primer-extension reaction with a proof-reading. non-displacing polymerase such as Pfu DNA polymerase. ‡ Select colonies with the correct DNA. Transform the circular nicked DNA into a highly competent strain such as XL1-Blue. ‡ Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make. These cells will repair the nicks and not restrict the unmodified product DNA.

Site-directed mutagenesis protocol .

Experimental protocol 1. Characterize the new enzyme . Verify production of the mutation 5. Pick target amino acid to be changed 2. Use this primer to synthesize double stranded DNA 4. Design a synthetic oligonucleotide to mutate the target amino acid 3.

see methods section for design tips) ± dH2O ± Dpn1 ± Competent cells .1ug/µl.Stratagene protocol This is the protocol for site-directed mutagenesis based on the stratagene kit ‡ Materials: ± Pfu turbo ± 10X Pfu turbo buffer ± dNTPs (10mM) ± Forward and reverse primers (0.

Methods For oligo-design or Primer Design: ‡ Both primers must contain the mutation. 5· 3· * 3· 5· . * ‡ The mutation should be in the middle of the primer. ‡ Primers should be 25-45 nucleotides long and have a GC content of at least 40%. ‡ The melting temperature (Tm) should be • 78ÛC. ‡ The 3¶-end of the primer has to end on an C or a G.

1 µg/ µl) dNTPs (10mM) Pfu turbo dH2O Amount 1 µl 5 µl 1µl 1 µl 1 µl 1 µl 40 µl .1 µg/ µl) Reverse Primer (0.Reaction ‡ Set up as follows: Components Template DNA (50ng/ µl) 10X Buffer Forward Primer (0.

this program can become very long. 680C for 7 minutes 4. or 18 cycles total 3. 600C for 50 seconds. 950C for 1 minute 2. 680C for 1 minute/kb of plasmid length ± repeat this step 17 times. 40C hold . so it may be best to turn overnight 1. 950C for 50 seconds.PCR program ‡ Depending on the length of the plasmid.

Incubate at 37 C for 1-2 hour to digest parental DNA ‡ Run 5µL of the digested reaction on a gel and compare to the undigested parental plasmid ± there should be some difference in band pattern.Following PCR DpnI Digest: ‡ Add 1 µl of DpnI to the reaction. Transformation: ‡ Transform the ¿nal reaction into competent cells .

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Discussion .

Trouble Shooting ‡ If no product is seen. . facilitating strand separation in GC rich regions of DNA and reducing the propensity of the DNA to form secondary structure. ‡ The end effect. ‡ DMSO disrupts base pairing. try repeating the protocol with 5% DMSO in the reaction mix. is a little DMSO will often get you past issues with poor primer design and/or dif¿cult templates.

Verifying the mutation Gene sequencing sequence the DNA to verify that the base changes have been introduced Restriction mapping use the creation of a new restriction site or the elimination of an existing one to verify the mutation .

Restriction screening Allows selection of mutant enzymes through the creation or elimination of a restriction endonuclease site The creation or elimination of a site changes the size of the DNA fragments obtained .

jove. 20010 from http://www.com/details. Nucl.php?id=1135 . 18. U Baumann. 2004 ‡ QuikChange® Site-Directed Mutagenesis Kit ‡ Retrieved May. 32:e115. and JL Reymond.References ‡ L Zheng. An ef¿cient one-step site-directed and sitesaturation mutagenesis protocol.. Acids Res.

Thank You for your kind attention! L/O/G/O www.com .themegallery.

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