User:Robwarden/Notebook/GFP-RGD Force Sensor/GFPst Plan

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Contents

Creating the GFP Expression Construct

Step 1: Amplify GFPuv

Primer Design

  • Forward Primer:
    • 5'-acgtacgt(Spacer)-tctaga(XbaI)-ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCC(Annealing)-3'
    • Tm: 66°C
    •  %GC: 42
  • Reverse Primer:
    • 5'-acgtacgt(Spacer)-cctgcagg(SbfI)-TTA(Stop)-CCCGGG(XmaI)-ATGATGGTGGTGATGGTG(His6)-CCGCGG(SacII)-GCCACCGCCGGTTTCCGGCAG(Sortase Tag)-GGAACCGCCACCACCAGAACCACCGCCGCCAGAGCCACCGCCACC(Linker)-TTATTTGTAGAGCTCATCCATGCCATGTGTAATCCC(Annealing)-3'
    • Tm: 65°C
    •  %GC: 42

Reaction Conditions

  • Phusion GC Buffer
  • Ta = 68°C
  • Extension Time = 30s

Step 2: Digestion/Ligation

  1. Digest both pMAL-c2x and PCR product
    • XbaI and SbfI
    • NEB Buffer 4 + BSA
    • Heat inactivate
  2. Incubate pMAL with Antarctic Phosphatase
    • Heat inactivate
  3. Quick Ligation

Step 3: Transformation

  • XL1-Blue Supercompetent
  • Amp plates
    • Add 40 μL IPTG to one plate (just to see if they glow)

Step 4: Validation

  • Digest Colonies with NcoI & BglII
    • NEB Buffer 3
    • Correct Pattern: 6465, 991
    • Empty Vector: 6645

Final Product

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