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

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

• File:PCRp-GFPst-Insert.gb
• Final Length: 847bp