User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/11/01

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PCR Based Mutagenesis Protocol

This is fast and efficient method for site directed mutagenesis using Type IIS restriction enzymes. With designing a pair of mutagenesis primers that the mutated sequence is near and downstream of the recognition site of the type IIs restriction enzyme. PCR will linearize the plasmid with two blunt ends. Enzyme digestion will produce two sticky and complementary ends that will ligate together and generate the mutated plasmid. Here this method would be applied to mutate the BSMBI cut site within the hygromycine resistant gene of V0200 mammalian vector. Primers are designed for SapI restriction enzyme:

PCR Mutagenesis.png

Here we plan to mutate the BSMBI cut site: CGTCTC to CGTTTC

Design Primers:

  • Forward Primer: 5' gca GCTCTTC G TTC + 25 bps of top right strand
  • Reverse Primer: 5' gca GCTCTTC G GAAACG + 25 bps of bottom left strand

PCR Reactions

Reagents Volume Reaction Conditions
DNA Template (ng) 200
  • 95°C, 3 min.
  • [95°C, 30 sec.; 45°C, 40 sec.; 72°C, * min (1 min/kb plasmid length)] x35
  • 72°C, 3 min.
  • 4°C, ∞
Forward Promer (µL) 1
Reverse Primer (µL) 1
2X GoTag (µL) 25
dH2O (µL) 22.8
Tolal (µL) 50

Run 5μL of each reaction on 1% agarose gel to confirm the plasmid linearization.

DNA Purification Using the GenElute™ PCR Clean-Up Kit for rapid purification of double-stranded PCR amplification products. Measure the DNA concentration with plate reader.

SapI Digestion

Cutting the SAPI sites to make the sticky ends for ligation:

Reagent Volume
DNA 500 ng
SapI μL 1.0
10X Buffer μL 5
dH2O μL 39.0
Total μL 50
Incubate at 37°C overnight

Restriction enzyme deactivation

Incubate the tubes at 80°C for 10 minutes and place the tube and heat block out of the heater on the bench cool down the digested parts gradually in room temperature.

Ligate the Sticky Ends to Make Circular DNA

DNA Conc. 10ng 20ng 30ng 50ng 50ng (Ctrl)
DNA 1μL 2μL 3μL 5μL 5μL
T4 Ligase 1.0 μL 1.0μL 1.0μL 1.0μL 0.0μL
2X Roche Buffer 5.00 μL 5.00 μL 5.00 μL 5.00 μL 5.00 μL
dH2O 5.00 μL 5.00 μL 5.00 μL 6.00 μL 0.00 μL
Total 10.0 μL 10.0 μL 10.0 μL 10.0 μL 10.0 μL
Incubate at room temperature for 30 minutes.

Bacterial transformation of each ligation reaction into DH5α-T , fast protocol: incubate DNA and cells on ice for 30 minutes and then spread on AMP agar plates and incubate in 37°C overnight.