Team:Cambridge/Protocols/PCRProtocol

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PCR using a high temperature DNA polymerase:

If this reaction is to be done from scratch, add the reagents in the order they are listed in the table. When multiple PCR experiments need to be run concurrently, everything that is not experiment specific (primers and template DNA) can be made up ahead of time as a master mix. In this case the master mix should be kept on ice as it contains enzymes. The primers and template DNA should be added to a PCR tube and the master mix added just before the sample is put in just before PCR begins.

Reagent	50 µl reaction	20 µl reaction	Final Concentration
H2O	Add to make 50 µl	Add to make 20 µl
5x Phusion buffer HF	10 µl	4 µl	1x
10 mM dNTPs	1 µl	0.4 µl	200 µM
Primer 1	x µl	x µl	0.5 µM
Primer 2	x µl	x µl	0.5 µM
Template DNA	x µl	x µl
Phusion DNA polymerase	0.5 µl	0.2 µl	0.02 u/ µl

N.B. The volume of primer that needs to be added will vary in accordance with the concentration of DNA in the initial sample.

PCR machine settings:

		Temperature (°c)	Time (s)
Hold 1		95	120
Cycle (30x)	Denaturing	98	10
	Annealing	60	30
	Elongation	72	120

Tips

Use touchdown PCR on reactions that show considerable mis-priming. Touchdown ensures that the first reactions are the most stringent in terms of primer annealing, so use of it ensures that the first reactions produce a pure product. Subsequent reactions then have very little additional template on which to mis-prime.
You can also use additional DMSO if you are having difficulties with mis-priming, as this disrupts secondary structures and so blocks the partial annealing of the primer to non-desirable sites.

Notes on Phusion use

Using the Tm calculator from [http://www.finnzymes.com/tm_determination.html Finnzymes], if annealing region is less than 20 bp, the annealing temperature should be the same as the highest calculated Tm of the various primers. Otherwise, it should be set to 3 °C above the highest Tm.

We have had successes designing our primers to have a Tm of around 60 °C and then having an annealing step of 58 °C.

Allow 15 - 30 seconds per kb for the elongation of product during the elongation step.

Do not exceed 1 min per kb of product during the elongation step.

Phusion is an excellent polymerase, but expensive. Use for large products and for products that require low error rates (e.g. production from tiny quantities of template).

Additionally, only Phusion has been tested in Gibson assembly. Do not substitute (unless you wish to further the sum total of human knowledge) during these reactions.

Notes on Velocity use

Using the [http://www.finnzymes.com/tm_determination.html Finnzymes] calculator, the annealing temperature should be between 2-5 °C below the lowest Tm of the primers being used.

Allow 15 - 30 seconds per kbp for the elongation step for templates up to 5kbp. Above this length, allow 1 min per kbp.

Risk Assessment Back to Protocols

@@ Line 48: / Line 48: @@
 *Using the Tm calculator from [http://www.finnzymes.com/tm_determination.html Finnzymes], if annealing region is less than 20 bp, the annealing temperature should be the same as the highest calculated Tm of the various primers. Otherwise, it should be set to 3 &deg;C above the highest Tm.
+*We have had successes designing our primers to have a Tm of around 60 &deg;C and then having an annealing step of 58 &deg;C.
 *Allow 15 - 30 seconds per kb for the elongation of product during the elongation step.