User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/10

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10/10/12

Site Directed Mutagenesis-Removing the BSMBI cut site from BD002 reporter gene

  1. Template strands are about 7kb for BD002.
  2. Forward primer: 0.15 mg and 32.6 nm, add 326μL dH2O to make 100μM and then diluted to 10μM concentration.
  3. Reverse primer: 0.16mg and 34.1 nm, add 341μL dH2O to make 100μM and then diluted to 10μM concentration.
  4. DNA template (BD002) miniprep concentration: 116ng/μL. Accuracy of the BD002 where checked with SapI and NotI cut.
  5. Serial dilution of template to: 1. 100 ng/μL 2. 80 ng/μL 3. 10 ng/μL 4. 1 ng/μL 5. 200 pg/μL
Reagents 1 2 3 4 5
Plasmid DNA 0.2 μL 0.5 μL 0.5 μL 0.5 μL 1 μL
primer 1 (10 μM, 125 ng) 1 μL 1 μL 1 μL 1 μL 1 μL
primer 2 (10 μM, 125 ng) 1 μL 1 μL 1 μL 1 μL 1 μL
2X GOTAG (PCR Master Mix) 25μL 25μL 25μL 25μL 25μL
dH2O 22.8 μL 22.5 μL 22.5 μL 22.5 μL 22 μL
Total 50 μL 50 μL 50 μL 50 μL 50 μL
  • No control reaction, I will test the accuracy of the mutagenesis with BSMBI restriction enzyme.
  • Thermal Cycling
    • 95°C/ 30 sec
    • [95°C/ 30 sec; 55°C/ 1 min; 72°C/ 6 min (1 min/kb plasmid length)]x30 cycle
    • 4°C, ∞
  • Run 5 μL of each PCR reaction on 10% agarose gel.
Alt text
Columns 1,3 amplified show BD002 ~6300 bp.
  • DNA Purification Using the GenElute™ PCR Clean-Up Kit for rapid purification of double-stranded PCR amplification products.
    • The purified result: Column1: 100ng/μL and Column 3: 28ng/μL.
  • Cutting the SAPI sites to make the sticky ends for ligation:
Amplified DNA (500ng) 5.00 μl 17.00 μL
SAPI 1.0 μl 1.0 μL
10x buffer 5.00 μl 5.00 μL
dH2O 39.00 μl 27.00 μl
Total 50.0 μl 50.0 μl
Incubate at 37°C overnight.
  • Restriction enzyme deactivation Incubate the tubes at 80°C for 10 minutes and cool down gradually in room temperature.
  • Ligate The Sticky Ends to Make Circular DNA
DNA Conc. 10ng 20ng 30ng 50ng 50ng (Ctrl)
DNA μL 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.
  • Long transformation protocol in BL-21.
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