Team:Colombia/Notebook/Protocols

From 2012.igem.org

(Difference between revisions)
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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.
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.
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==='''Primer Design'''===
==='''PCR - Pfu DNA polimerase'''===
==='''PCR - Pfu DNA polimerase'''===
-
One reaction
+
One reaction.
{|
{|
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|-
|-
|H2O
|H2O
-
|16.95
+
|17.95
|-
|-
|Buffer
|Buffer
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|-
|-
!Total
!Total
-
|10
+
|25
|}
|}
==='''PCR - Invitrogen (R) Taq polimerase'''===
==='''PCR - Invitrogen (R) Taq polimerase'''===
 +
 +
One reaction.
 +
 +
{|
 +
! align="left"| 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
 +
|-
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!Total
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|10
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|}
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 +
If needed add 1 μL of DMSO per reaction, and adjust the amount of H2O to a final volume of 10μL.

Revision as of 22:10, 13 July 2012

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 - Invitrogen (R) Taq 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.

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