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From 2010.igem.org

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Revision as of 20:53, 27 October 2010

MAIN STEPS/ TIME TABLE
- Each step is carried out for 1 tube
- PCR solution preparation [30-45 min]
- Thermocycler [2.5 h]

   
- Denaturation
   
- Annealing
   
- Primer extension

- PCR purification [30 min]
- DNA concentration determination with Nanodrop [5 min]
- Electrophoresis [2 h]

MATERIALS
- Thermal Cycler [M08-Techne TC-512 Gradient PCR]
- Spin [M03]
- P10 and P100 micropipetes
- DNase-free micropipet tips
- Microfuge tubes (0,5 mL, thin-walled for amplification reactions)
- Electrophoresis [MO6]
- Nanodrop [M08]

- PCR kit (MasterMix)
- Forward primer
- Reverse primer
- Template DNA
- Nuclease-free water
- PCR purification kit

SOLUTIONS
- PCR MasterMix Kit [Fermentas]

CHECK-LIST PROCEDURE
Sample Preparation
- Calculate the amount of your samples.
- Design the tubes.
- Label the microfuge tubes.
- Transfer the PCR reaction to a pre-heated block.

- Adjustment of thermocycler [changeable parameters]
- Lid Temp: 105 C (to prevent evaporation)
- Step 1 (initial denaturation): 94 C, 5 min. Press select to go.
- Step 2 (cycle denaturation): 94 C, 30 sec.
- Step 3 (cycle annealing): primer specific, 30 sec.
- Step 4 (cycle extension): 72 C, 1 min. Press select two times.
- Step 5 (cycle loop): Go to step 2, 30 to 35 cycles.
- Step 6 (final extension): 72 C, 15 min.
- Step 7 (store): 4 C, forever.

Follow-Up Steps
(It is recommended that you use PCR products for RE digestion and ligation in the same day to prevent the damage to DNA ends)

Agarose gel electrophoresis
- PCR product purification is NOT needed for gel loading. You can directly load the PCR product to check the results.

NOTES
- To reduce the chance of contamination with exogenous DNAs, bake all glassware for 6 h at 15 C and autoclave all plasticware.
- If the thermal cycler is not equipped with a heated lid, use either mineral oil or paraffin wax to prevent evaporation of liquid from the reaction mixture.
- High concentrations of of dNTPs (> 4mM) are inhibitory, because of sequestering Mg2+.
- Pfu is more suitable, error rate of it per nucleotide is the lowest of any DNA polymerase.
- Inhibition of PCR can be caused by proteinase K, phenol, EDTA, ionic detergents, heparin, gel-loading dyes (bromophenol blue, xylene cyanol)

TROUBLESHOOTING
No bands
- PCR kit or polymerase enzyme is not working: Include positive control (ex. a control DNA sequence with primers)
- Primers are not working

   
- Secondary structure: Use DMSO for helping denaturation of DNA secondary structures
   
- Melting temperatures are different: see annealing temperature 
   
- Annealing temperature is not right: Use 50 C. It is used as a generic temperature by sequencing center to amplify all different samples.

- Problem with template and primer working solutions:

   
- Measure concentration. 
   
- Include positive control (ex. a control DNA sequence with primers)
   
- Increase the amount of template

Non-specific bands
- If you see no PCR product then decrease annealing temp 5-10 C; if you see non-specific products, then increase it by 5-10 C.

Alternative Procedure
- Master Mix

   
- 3 uL-10X Taq Buffer
   
- 3 uL-2mM dNTP mix
   
- 3 uL-25mM MgCl2
   
- 18 uL Nuclease-free water

- For 30 uL PCR mix

   
- 27 uL Master mix
   
- 3 uL-5uM forvard primer
   
- 0.25 uL-5 u/uL Taq
   
- 3 uL-250 ng/uL Template

Alternative Procedure with Pfu DNA polymerase
- 10 uL 10X Pfu Buffer
- 8 uL dNTPs (2.5 mM stock)
- 2.5 uL primer 1 (100 ng/uL)
- 2.5 uL primer 2 (100 ng/uL)
- 1 uL Pfu enzyme
- x uL template ( around 10 ng)
- Complete to 100 uL with nuclease-free water

- 94 C 1 min
- 53 C 1 min
- 72 C 1 min 30 sec for 30 cycles
- End on a 10 min 72 C extension and keep at 4 C.

- Extension times are longer for Pfu than Taq.
- If amplification does not work for first time, add extra MgCl2.