User:Michaela Harper/Notebook/Biological Chemistry Lab/2011/09/20
Biomaterials Design Lab | Main project page Previous entry Next entry |
ObjectivePerform PCR to mutate GFP to contain a cysteine residue just after the enterokinase cleavage site using protocols found in Stratagene Quick Change PCR manual DescriptionMaterials GFP plasmid sequence from Invitrogen
10× Reaction Buffer: 100 mM KCl 100 mM(NH4)2SO4 200 mM Tris-HCl (pH 8.8) 20 mM MgSO4 1% Triton X-100 1 mg/ml nuclease-free bovine serum albumin (BSA)
SAMPLE: 5 μl of 10× reaction buffer X μl (5–50 ng) of dsDNA template X μl (125 ng) of oligonucleotide primer #1 X μl (125 ng) of oligonucleotide primer #2 1 μl of dNTP mix ddH2O to a final volume of 50 μl Then add: 1 μl of PfuTurbo DNA polymerase (2.5 U/μl)
Temperature cycling: Segment 1: 1 cycle, 95 degrees, 30 seconds Segment 2: 16 cycles, 95 degrees, 30 seconds 55 degrees, 1 minute 68 degrees, 3.6 minutes (for 1min/kb of plasmid, 3600 bps of plasmid) Leave reaction mixture in thermocycler at 4°C for 24 hr. DataDetermined by Group 3: Forward Primer: GAT AAG GAT GAC GAT AAG TGT CGA TGG GGA TCC GAA TTC GCC Reverse Primer: GGC GAA TTC GGA TCC CCA TCG ACA CTT ATC GTC ATC CTT ATC Mutation in bold. 39bp, 51% GC content, Tm=78.8°C (salt adjusted),
Actual primers used: Forward Primer: 5'- GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA -3' Reverse Primer: 5'- TTC GGA TCC CCA TCG ATC CTT ATC GTC ATC GTC- 3' Notes
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