实验概要

protocal for PCR

实验步骤

PCR

1) Add the following to a microfuge tube:
        10 ul reaction buffer
        1 ul 15 uM forward primer
        1 ul 15 uM reverse primer
        1 ul template DNA
        5 ul 2 mM dNTP
        8 ul 25 mM MgCl2 or MgSO4 (volume variable)
                     water (to make up to 100 ul)

2) Place tube in a thermocycler. Heat sample to 95 °C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.

3) Start the PCR cycles according the following schemes:

        a) denaturation - 94 ° C, 30-90 sec.
        b) annealing - 55 °C (or -5° Tm), 0.5-2 min.
        c) extension - 72 °C, 1 min. (time depends on length of PCR product and enzyme used)
                         repeat cycles 29 times

4) Add a final extension step of 5 min. to fill in any uncompleted polymerisation. Then cooled down to 4- 25 °C.

注意事项

a) Mg - one of the main variables - change the amount added if the PCR result is poor. Mg affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg can change the Mg concentration.

b) Template DNA concentration - PCR is very powerful tool for DNA amplification therefore very little DNA is needed. But to reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used, though too much template may increase the amount of contaminants and reduce efficiency.

c) Enzymes used - Taq DNA polymerase has a higher error rate (no proof-reading 3' to 5' exonuclease activity) than Tli or Pfu. Use Tli, Pfu or other polymerases with good proof-reading capability if high fidelity is needed. Taq, however, is less fussy than other polymerases and less likely to fail. It can be used in combination with other enzymes to increase its fidelity. Taq also tends to add extra A's at the 3'end (extra A's are useful for TA cloning but needs to be removed if blunt end ligation is to be done). More enzymes can also be added to improve efficiency (since Taq may be damaged in repeated cycling) but may increase non-specific PCR products. Vent polymerase may degrade primer and therefore not ideal for mutagenesis-by-PCR work.

d) dNTP - can use up to 1.5 mM dNTP. dNTP chelate Mg, therefore amount of Mg used may need to be changed. However excessive dNTP can increase the error rate and possibly inhibits Taq. Lowering the dNTP (10-50 uM) may therefore also reduce error rate. Larger size PCR fragment need more dNTP.

e) primers - up to 3 uM of primers may be used, but high primer to template ratio can results in non-specific amplification and primer-dimer formation (note: store primers in small aliquots).

f) Primer design - check primer sequences to avoid primer-dimer formation. Add a GC-clamp at the 5' end if a restriction site is introduced there. One or two G or C at the 3' end is fine but try to avoid having too many (it can result in non-specific PCR products). Perfect complementarity of 18 bases or more is ideal. See Guide.

g) Thermal cycling - denaturation time can be increased if template GC content is high. Higher annealing temperature may be needed for primers with high GC content or longer primers (calculate Tm). Using a gradient (if your PCR machine permits it) is a useful way of determining the annealing temperature. Extension time should be extended for larger PCR products; but reduced it whenever possible to limit damage to enzyme. Extension time is also affected by the enzymes used e.g for Taq - assume 1000 base/min (also check suppliers' recommendations, actual rate is much higher). The number of cycle can be increased if the number of template DNA is very low, and decreased if high amount of template DNA is used (higher template DNA is preferable for PCR cloning - lower error rate in the PCR).

h) Additives -

1. Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). Glycerol can protects Taq against heat damage, while formamide may lower enzyme resistence.
   

2. 0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC content and long stretches of DNA (Long PCR / LA PCR). Perform a titration to determine to optimum concentration (1.3 M recommended). Reduce melting temperature (92 -93 °C) and annealing temperature (1-2°C lower). It may be useful to use betaine in combination with other reagents like 5%DMSO. Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits.
   

3. >50mM TMAC (tetramethylammonium chloride), TEAC (tetraethylammonium chloride), and TMANO (trimethlamine N-oxide) can also be used.

4. BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction.

5. See also Dan Cruickshank's PCR additives and Alkami Enhancers for more.

i) PCR buffer

1. Higher concentration of PCR buffer may be used to improve efficiency.
   

2. This buffer may work better than the buffer supplied from commercial sources.
   
   16.6 mM ammonium sulfate
   67.7 mM TRIS-HCl, pH 8.89
   10 mM beta-mercaptoethanol
   170 micrograms/ml BSA
   1.5-3 mM MgCl2