Maheshri:PCR

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PCR

Narendra Maheshri

January 2007

Components

A PCR reaction consists of the following components:

1. Polymerase: We use either Taq DNA polymerase or Expand, a mixture of Taq and Pfu. The Pfu DNA polymerase has much higher fidelity (due to the 3’->5’ exonuclease activity) so Expand is often the reagent of choice when the PCR product will be used for gene replacement, sequencing, or cloning. For check PCR, Taq is fine. Our Taq and Expand working stocks have been diluted to be used at 100X.
2. Thermopolymerase buffer: The recipe below has been used successfully with Taq, Pfu, and Vent, and can probably be used with other polymerases. It is typically made at 10X.
3. Magnesium: Mg2+ is used as a cofactor by the DNA polymerase. Its concentration can have significant effects on the outcome of the PCR and is often varied when a PCR reaction needs optimization. We use a 10X stock solution of 20 mM MgCl2, such that the final [Mg2+] is 2 mM. For optimization, typically a range of [Mg2+] from 1.5 – 4.5 mM is used. Additional Mg2+ often increases yields but decreases selectivity/specificity.
4. dNTP’s; The monomers of the PCR reaction are bought separately, and all 4 must be mixed at equal ratios. We make 2 mM dNTP stocks (that means EACH dNTP is at 2 mM) and freeze at -20C. This can be used at 10X for a final dNTP concentration of 0.2 mM.
5. Primers: Primers should be used at a final concentration of anywhere from 0.1-2 μM. A good starting point is ~ 0.5 μM. Primer stocks are typically diluted to >10 μM (often 100 μM) in TE. Make working stocks by diluting to 10 mM in milliQ H2O. A crucial parameter for optimization is the primer:template ratio. When there is a lot of non-specific template (ie in the case of PCR using genomic DNA as template), using higher primer concentrations often yield better results.
6. Template: When doing PCR using either plasmid or genomic DNA as template, template concentrations in the range of 5-50 ng/μL. For example, you can use a miniprep as 100X in your PCR reaction. Too much template is a bad thing and results in template inhibition (mostly linear amplification rather than exponential). Often times varying template amount can be used for optimization.
7. DMSO: DMSO and glycerol are often used as additives (up to 10 % v/v) to lower the effective melting temperature. If you are having difficulty getting a PCR to work, especially if the amplicon is very long and/or GC rich, try adding DMSO to 5 or 10% v/v.
8. Reaction volume: PCR reactions do not always scale up appropriately: what works at 50 μL may not work at 100 uL reaction volume. During optimization, I often use a 20 μL reaction volume to find the right condition. If I need more PCR product (say for yeast transformation) I scale out rather than scale up, and do 4 x 50 μL reactions.

Conventional PCR Protocol

Recipe

Below is a recipe for 2 x 50 μL PCR reactions

10 μL		10x ThermoPol buffer
10 μL		20 mM MgCl2 	
10 μL		2 mM dNTP mix
4  μL		sense primer (10 μM)
4  μL		antisense primer (10 μM)
1  μL          template (~ 100 ng/uL)
1  μL		Taq or Expand
60 μL		water 			

Alternatively, if the template used for each PCR reaction were different, this mix could be made using everything EXCEPT the template, and then 49 μL could be added to two PCR reaction tubes containing 1 μL of each template.

Running the reaction

You want to make sure the PCR reaction does NOT start at room temperature when the primers and template may anneal to each other in a non-specific way. Otherwise any non-specific PCR product created will be used as template for your reaction!

Tips;

• Do everything on ICE

• Add the polymerase last when you can, otherwise add the template last

• Do a hotstart – transfer the tubes from the ice directly to a PRE-HEATED PCR block (should be at > 90°C)

Standard cycling

A simple cycling protocol is given below

Initial 2 min @ 94C

Cycle: 20-35 times (see below for Number of Cycles)

0.5 - 1 min @ 94C

0.5 - 1 min at 45-70C (see below for Hybridization Temperature)

1-5 min at 72C (see below for Polymerization Time)

Final 10 min @ 72C

Place in refrigerator, or set PCR cycler to 10C forever.

The Number of Cycles depends on the application. For subcloning from a plasmid template, it is best to run a lower number of cycles (20-30) from a higher quantity of template (>=50 ng). For RT-PCR, a high number of cycles (>=30) may be necessary to visualize a product. For quantitative RT-PCR, the number of cycles must be optimized to ensure that the reaction is within the linear amplification range.

The Hybridization Temperature, which determines the stringency of the PCR, is guided by the oligonucleotides’ melting temperature (Tm). Tm can be roughly calculated by the formula Tm =2*(A or T content) + 4*(G or C content), and the hybridization temperature can be set about 5C below Tm. This is critical for getting specific products from PCR from cDNA, and care must be taken to design primes with high Tm. For subcloning from plasmids, where there is only one template, the hybridization temperature can be set relatively low, 45-50C. PCR stringency can also be varied with the magnesium concentration.

The Polymerization Time is generally 1 minute per each kb of product length.

Touchdown cycling

Oftentimes, a touchdown PCR is desirable when the primers used have large overhangs. Consider a PCR reaction to amplify a product to be used for gene replacement in yeast. 20 bp of each primer hybridize to the template and 40-50 bp do not. Here, the first few cycles are done at a low hybridization temperature to ensure the 20 bp of each primer anneals to the template and results in a PCR product. But after that, the hybridization temperature is brought way up since now 60-70 bp are hybridizing to the template at each round of amplification. Here is a typical protocol:

Initial 2 min @ 94C

Cycle: 10 times (see below for Number of Cycles)

1 min @ 94C

1 min at 45-55C

1-5 min at 72C

Cycle 20 times

0.5 min @ 94C

1-5 min @ 72C

Final 10 min @ 74C

Notice here the hybridization temperature IS the extension temperature.

PCR Precipitation

For cleanup of the reaction mixture following PCR, use Qiagen's QIAquick PCR kit or the following isopropanol precipitation protocol.

• Following PCR, add 4 parts IPA and 1 part 3M NaAc to 5 parts PCR mixture. Incubate at -80°C for 30'.
• Spin down for 10' at max speed.
• Aspirate the supernatant and wash pellet with 70% EtOH. Spin 5'.
• Aspirate the EtOH and dry the pellet.
• Finally, resuspend in 20μL ddH₂O.