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From 2012.igem.org

Contents 1 Protocols 1.1 Bacterial DNA extraction protocol 1.2 Miniprep 1.3 Electroporation 1.4 Primer Design 1.5 PCR - Pfu DNA polimerase 1.6 PCR - Taq DNA polimerase 1.7 PCR - Colony 1.8 PCR - Boiling

Protocols

Bacterial DNA extraction protocol

Miniprep

Electroporation

Electro-competent cells of E. coli DH5α are electroporated at 1.25 mV, immediately we add SOC medium to the bacteria and we incubated on a shaker at 37°C for one hour, then, we culture cells in a solid medium containing the antibiotic of selection.

Primer Design

PCR - Pfu DNA polimerase

One reaction.

Reactives	Amount (μL)
H2O	17.95
Buffer	2
MgSO4	1.6
dNTP's	0.4
Primer Fw	0.4
Primer Rv	0.4
Taq	0.06
Pfu	0.19
DNA	2
Total	25

PCR - Taq DNA polimerase

One reaction.

Reactives	Amount (μL)
H2O	6.15
Buffer	1
MgCl2	0.5
dNTP's	0.25
Primer Fw	0.5
Primer Rv	0.5
Taq	0.1
DNA	1
Total	10

If needed add 1 μL of DMSO per reaction, and adjust the amount of H2O to a final volume of 10μL.

PCR - Colony

Based on the protocol used with Taq polimerase, add 1μL more of H2O and take a few cells from the strain pricking it with a toothpick and mixing them with the other PCR reactives instead of using purified DNA.

PCR - Boiling