3 Cloning of a region of interest into a plasmidPlasmidRegion of interestChoose the right vector (+/- tag,and if tagged, a C- or N-terminal tag).Blunt end or sticky end cloning.Check that the enzymes chosen inthe MCS for the cloning don’t cutin the region of interest.Check that there is enough “space”in-between the two enzymes in the MCS.Sticky endsMCSBlunt ends
4 PrimersCheck the reading frame.C-terminal tag: Add a Kozak consensus sequence (ANNATGG) if it is a C-terminal tag, and again remember to clone the gene in frame with the C-terminal tag. Don’t forget to take the STOP codon out.Choose enzymes in the MCS to add to the primer that do not cutin the insert.Primers should be nucleotides long and have a GC content of %.The forward and reverse primer should have similar melting temperatures (Tm’s).Try to avoid primer dimer and hairpin formation.For sticky end ligation remember to add additional nucleotides to the 5’ side of the restriction site. The restriction site sequences should be followed by 15 bases that are homologous to the template DNA.The 3’-end of the primer has to end on an C or a G.
6 The purpose of the PCR is to make a huge number of The principle of the PCR (Roche)The purpose of the PCR is to make a huge number ofcopies of a gene of interest3 steps in a PCR repeated for cycles.Denaturation at 95˚C: the double stranded DNA melts open to single stranded DNA.Annealing/hybridisation: The primers anneal to the DNA.Extension 68˚C or 72˚C: The polymerase copies the template adding the dNTPsfrom 5’ to 3’.
7 PCR 0.1-5 ng plasmid DNA and 0.1-1 ug for genomic DNA 200 µM dNTPs (final concentration)1-1.5 U Taq polymerase and Pfu Turbo polymerase U0.1-1 µM of each primer fwd/rev1-4 mM MgCl2 Taq polymerase (MgSO4 for Pfu polymerase)DMSO up to 10% (has been shown to facilitate DNA separation)Initial denaturation: 1 Cycle: 1 min. 95˚C is usually enough for plasmid DNA. For genomic DNA it will have to be longer.30-40 cycles PCR.If using primers with restriction sites use a lower annealing temperature for the first 10 cycles of the PCR.Final extension step: Incubate samples at 68˚C /72˚C for 5-15min to fill in the protruding ends of the PCR products. Taq DNA Polymerase adds extra A nucleotides to the 3'-ends of PCR products. If PCR fragments are cloned into T/A vectors, this step can be prolonged to up to 30min.5’3’
8 CloningDigest the vector and the amplified DNA for 2h or O.N. depending on the enzymes used.Clean up the digested vector and fragment.Check the concentration of the vector and the fragment on a gel.For Blunt end ligation (ratio vector : fragment; 1pmol : 10pmol)For sticky end ligation (ratio vector : fragment; 1pmol : 3pmol)Negative control with vector only.
9 Problems after ligation Colonies on the negative control.Poor cutting of the vector by the restriction enzymes. De-phosphorylation of the vector (also if using two different enzymes).Colony screening using PCR.No colonies:Poor cutting of the PCR product by the restriction enzyme because of inefficient extension by the polymerase.Poor cutting due to insufficient number of extra nucleotides at the 5’ side of the restriction site. Clone the fragment blunt end and then cut it out.Blunt end ligation: Use PfuTurbo polymerase.Taq DNA Polymerase adds extra A nucleotides to the 3'-ends of PCR products. Clone the PCR product into a A/T cloning vector or treat it with Klenow and dNTPs and then ligate.
10 Site-directed mutagenesis Workflow (Stratagene)Gene in plasmid with target site mutationDenature the plasmid and anneal the oligonucleotideprimers containing the desired mutationUsing the non-strand-displacing action of PfuTurbopolymerase, extend and incorporate the mutagenicprimers resulting in nicked circular strandsDigest the methylated, nonmutated parental DNAtemplate with Dpn ITransform the circular, nicked dsDNA into super-competent cellsAfter transformation the supercompetent cellsrepair the nicks in the mutated plasmid
11 site-directed mutagenesis Primer design forsite-directed mutagenesisStratagene:Both primers must contain the mutation.The mutation should be in the middle of the primer.Primers should be nucleotides long and have a GC content of at least 40%.The melting temperature (Tm) should be ≥ 78˚C.The 3’-end of the primer has to end on an C or a G.5’3’*
12 site-directed mutagenesis Primer design forsite-directed mutagenesisInvitrogen:Only one primer contains the mutation.Both primers should be ~ 30 nucleotides long.Primers should have an overlapping region at the 5’ end of nucleotides.On the mutagenic primer, there should be at least 10 nucleotidesdownstream of the mutation.Literature:(Zheng et al. An efficient one-step site-directed and site-saturationmutagenesis protocol. Nucleic Acids Res Aug 10;32(14):e115).Both primers must contain the mutation.At least 8 non-overlapping bases at the 3’ end.The 3’-end of the primer has to end on an C or a G.5’3’*5’3’*
13 Site-directed mutagenesis 5-50 ng/50 µl reactions of dsDNA plasmid DNA from a dam+ E.coli strainng/50 µl reactions of forward and reverse primer (PAGE or HPLC purified)200 µM dNTPs2 mM MgSO42.5 U of PfuTurbo polymeraseDMSO up to 10% (has been shown to facilitate DNA separation)Problems:No product: Don’t continue.When using DMSO add more PfuTurbo polymerase.If the oligo anneals several times, extend the oligo and increase theannealing temperature.Primer dimer and hairpin: Use asymmetric oligos.Repeats on either site of the mutation: Try shorter oligos and increasethe annealing temperature
14 Papers and useful websites PCRBarnes WM. PCR amplification of up to 35-kb DNA with high fidelityand high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A.1994 Mar 15;91(6):Site-directed mutagenesisZheng et al. An efficient one-step site-directed and site-saturationmutagenesis protocol. Nucleic Acids Res Aug 10;32(14):e115.
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