Team:Colombia/Notebook/Protocols

From 2012.igem.org

Revision as of 22:26, 13 July 2012 by Gutiloluis (Talk | contribs)

Contents

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