|
|
Line 660: |
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| 2. Annealing : temperature is lowered to enable the DNA primers to attach to the template DNA <br> | | 2. Annealing : temperature is lowered to enable the DNA primers to attach to the template DNA <br> |
| 3. Extending : temperature is raised and the new strand of DNA is made by the polymerases <br></div> | | 3. Extending : temperature is raised and the new strand of DNA is made by the polymerases <br></div> |
− | Thermocycling Conditions for a Routine PCR: | + | Thermocycling Conditions for a Routine PCR: <br> |
− | STEP
| + | <table> |
− | TEMP
| + | <tr> |
− | TIME
| + | <th align="center"><b>Step</b></th> |
− | Initial Denaturation | + | <th align="center"><b>Temp</b></th> |
− | 98°C | + | <th align="center"><b>Time</b></th> |
− | 30 seconds | + | </tr> |
− | 25–35 Cycles
| + | <tr> |
− | 98°C | + | <td align="center"><b>Initial Denaturation</b></td> |
− | 5–10 seconds | + | <td align="center">98°C</td> |
− | *50–72°C
| + | <td align="center">30 seconds </td> |
− | 10–30
| + | </tr> |
− | seconds
| + | <tr> |
− | 72°C
| + | <td align="center"><b></b></td> |
− | 20–30
| + | <td align="center">98°C</td> |
− | seconds
| + | <td align="center">5–10 seconds</td> |
− | /kb | + | </tr> |
− | Final Extension
| + | <tr> |
− | 72°C
| + | <td align="center"><b>25–35 Cycles</b></td> |
− | 2
| + | <td align="center">*50–72°C</td> |
− | minutes
| + | <td align="center">10–30 seconds</td> |
− | Hold
| + | </tr> |
− | 4–10°C
| + | <tr> |
− | hold is not
| + | <td align="center"><b></b></td> |
− | necessary
| + | <td align="center">72°C</td> |
− | 1.
| + | <td align="center">20–30 seconds/kb</td> |
− | Template:
| + | </tr> |
− | Use of high quality, purified DNA templates greatly enhances the success of PCR.
| + | <tr> |
− | Recommended amounts of DNA template for a 50 μl reaction are as follows:
| + | <td align="center"><b>Final Extension</b></td> |
− | DNA
| + | <td align="center">72°C</td> |
− | AMOUNT
| + | <td align="center">2 minutes</td> |
− | DNA Genomic
| + | </tr> |
− | 1 ng–1 μg
| + | <tr> |
− | Plasmid or Viral
| + | <td align="center"><b>Hold</b></td> |
− | 1 pg–1 ng
| + | <td align="center">4-10°C</td> |
− | 2.
| + | <td align="center"></td> |
− | Primers:
| + | </tr> |
− | Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC
| + | </table> |
− | content of 40–60%. Computer programs such as
| + | |
− | Primer3
| + | |
− | can be used to design or analyze | + | |
− | primers. The best results are typically seen when using each primer at a final concentration
| + | |
− | of 0.5 μM in the reaction.
| + | |
− | 3.
| + | |
− | Mg
| + | |
− | ++
| + | |
− | and additives:
| + | |
− | The
| + | |
− | Q5 High-Fidelity Master Mix contains 2.0
| + | |
− | mM Mg
| + | |
− | ++
| + | |
− | when used at a 1X concentration.
| + | |
− | This is optimal for most PCR products generated with this master mix.
| + | |
− | 4.
| + | |
− | Deoxynucleotides:
| + | |
− | The final concentration of dNTPs is 200 μM of each deoxynucleotide in the 1X
| + | |
− | Q5 High-
| + | |
− | Fidelity Master Mix.
| + | |
− | Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not
| + | |
− | recommended for use with uracil-containing primers or templates.
| + | |
− | 5.
| + | |
− | Q5
| + | |
− | High-Fidelity DNA Polymerase concentration:
| + | |
− | The concentration of
| + | |
− | Q5 High-Fidelity DNA Polymerase in the
| + | |
− | Q5 High-Fidelity 2X Master
| + | |
− | Mix has been optimized for best results under a wide range of conditions.
| + | |
− | 6.
| + | |
− | Denaturation:
| + | |
− | An initial denaturation of 30 seconds at 98°C is sufficient for most amplicons from pure
| + | |
− | DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that
| + | |
− | require it.
| + | |
− | During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10
| + | |
− | second denaturation at 98°C is recommended for most templates.
| + | |
− | 7.
| + | |
− | Annealing:
| + | |
− | Optimal annealing temperatures for
| + | |
− | Q5 High-Fidelity DNA Polymerase tend to be higher
| + | |
− | than for other PCR polymerases. The
| + | |
− | NEB T
| + | |
− | m
| + | |
− | Calculator | + | |
− | should be used to determine the
| + | |
− | annealing temperature when using this enzyme. Typically use a 10–30 second annealing step
| + | |
− | at 3°C above the T
| + | |
− | m
| + | |
− | of the lower T
| + | |
− | m
| + | |
− | primer. A temperature gradient can also be used to
| + | |
− | optimize the annealing temperature for each primer pair.
| + | |
− | For high T
| + | |
− | m
| + | |
− | primer pairs, two-step cycling without a separate annealing step can be used
| + | |
− | (see note 10).
| + | |
− | 8.
| + | |
− | Extension:
| + | |
− | The recommended extension temperature is 72°C. Extension times are generally 20–30
| + | |
− | seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for | + | |
− | simple templates (plasmid,
| + | |
− | E. coli
| + | |
− | , etc.) or complex templates < 1 kb. Extension time can be
| + | |
− | increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.
| + | |
− | A final extension of 2 minutes at 72°C is recommended.
| + | |
− | 9.
| + | |
− | Cycle number:
| + | |
− | Generally, 25–35 cycles yield sufficient product.
| + | |
− | For genomic amplicons, 30-35 cycles are
| + | |
− | recommended.
| + | |
− | 10.
| + | |
− | 2-step PCR:
| + | |
− | When primers with annealing temperatures ≥
| + | |
− | 72°C are used, a 2-step thermocycling protocol
| + | |
− | (combining annealing and extension into one step) is possible.
| + | |
− | 11.
| + | |
− | Amplification of long products:
| + | |
− | When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50
| + | |
− | seconds/kb.
| + | |
− | 12.
| + | |
− | PCR product:
| + | |
− | The PCR products generated using
| + | |
− | Q5 High-Fidelity
| + | |
− | 2X
| + | |
− | Master Mix
| + | |
− | have blunt ends. If
| + | |
− | cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred,
| + | |
− | the DNA should be purified prior to A-addition, as
| + | |
− | Q5 High-Fidelity DNA Polymerase will
| + | |
− | degrade any overhangs generated.
| + | |
− | Addition of an untemplated -dA can be done with
| + | |
− | Taq
| + | |
− | DNA Polymerase (
| + | |
− | NEB #M0267
| + | |
− | ) or
| + | |
− | Klenow exo
| + | |
− | –
| + | |
− | (
| + | |
− | NEB #M0212
| + | |
− | )
| + | |
| <br> <br> | | <br> <br> |
| Please note that protocols with | | Please note that protocols with |