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

=Creating the GFP Expression Construct=

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
 * 2) *XbaI and SbfI
 * 3) *NEB Buffer 4 + BSA
 * 4) *Heat inactivate
 * 5) Incubate pMAL with Antarctic Phosphatase
 * 6) *Heat inactivate
 * 7) 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

 * [[Image:PCRp-GFPst-Insert.gb|Product File]]
 * Final Length: 847bp