User:Michaela Harper/Notebook/Biological Chemistry Lab/2011/09/20

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Objective

Perform PCR to mutate GFP to contain a cysteine residue just after the enterokinase cleavage site using protocols found in Stratagene Quick Change PCR manual

Description

Materials GFP plasmid sequence from Invitrogen


  • Design forward and reverse primers for the plasmid in order to meet the requirements laid out in the Quick Change Manual
    • A "calculator" to determine the melting temperature of oligonucleotides can be found here. When performing the calculation leave out the nucleotide bases that you are mutating. (These bases are going to be mismatched during the PCR runs).
    • Primers must have a melting point, Tm ≥ 78°C, a GC content ≥ 40%, and be 25-45 bps in length.
  • Figure out what solutions you will need to perform a PCR run and what volumes of each solution you will need. Check on the stock solutions to make these calculations.

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)

  1. Prepare reaction mixture with 5 μl of 10× reaction buffer, 1 μL of dsDNA template(50 ng/μL), 1.25 μL of oligonucleotide control primer #1 (100 ng/μL), 1.25 μl of oligonucleotide control primer #2 (100 ng/μL), 1 μl of dNTP mix (10mM), and 41.75 μL of double-distilled water (ddH2O) to a final volume of 50 μL.
  2. Add 1 μL of PfuTurbo DNA polymerase (2.5 U/μL) to the reaction mixture.
  3. Add 25 μL Bio Rad Chill Out™ liquid wax.
  4. Design a temperature cycling program for the thermocycler.

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.

Data

Determined 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

PCR song

protein translation

protein translation

inner life of a cell




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