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From 2012.igem.org

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Contents 1 PCR with Phusion High-Fidelity DNA Polymerase 1.1 Tube preparation 1.2 Cycling instructions 2 Préparation of LB medium and LB Agar: 3 Gel extraction

PCR with Phusion High-Fidelity DNA Polymerase

Tube preparation

Put items in this order:

Component	50µl reaction	Comments
H2O	32
5x Phusion HF Buffer	10
10mM dNTPs	1
Primer FW	2	Primers have to be at 10µM
Primer RV	2	Primers have to be at 10µM
Template DNA	1
DMSO (optional)	1,5	recommended for GC-rich amplicons < 20kb
Phusion DNA polymerase	0,5

Cycling instructions

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	4min	1
Denaturation	98°C	20s	30
Annealing	Lower Tm of primers	30s
Extension	72°C	30S/kb
Final extension	72°C	10min	1
	4°C	hold

Préparation of LB medium and LB Agar:

=> LB Agar : -18,5g LB Agar -300ml H2O

=> LB medium : -6g LB broth -300ml de H2O

Autoclaved at 250°C

Gel extraction

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel under UV light. 2. Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel. 3. Incubate at 50°C for 10 min, until the gel slice has completely dissolved. 4. The color of the mixture have to be yellow, otherwise add 10 µl of 3M sodium acetate. 5. Ass 1 gel volume of isopropanol to the sample and mix. 6. Place a spin column in a provided 2 ml collection tube. 7. To bind DNA, apply the sample to the column and centrifugat for 1 min. Discard the flow-through and place the column back into the same tube. 8. To wash, add 0,75 ml of buffer PE to the column and centrifugate for 1 min.Discard the flow-through and place the column back into the same tube. 9. Centrifugate the column in a 2 ml Collection tube for 1 min 17,900xg (13,000 rpm). 10. Place the column into a clean 1,5 ml microcentrifuge tube. 11. to elute DNA, add 50 µl of water to the column and centrifugate for 1 min.

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