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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 1 μL 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

We make a lysate of the cells and add 1 μL of that lysate instead of purified DNA using the Taq polimerase protocol. To obtain the lysate, scrape the strain and resuspend the cells taken on 50 μL of H2O, then, put at 95°C for 10 minutes.