Team:Amsterdam/Notebook/Protocols/Ligations

From 2011.igem.org

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=Ligation=
=Ligation=
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After following our digestion protocol a ligation can be performed.<br>
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After following our [https://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Digestion digestion protocol] a ligation can be performed.<br>
Different ratios of vector:insert are used, to get the best result out of the ligation.<br>
Different ratios of vector:insert are used, to get the best result out of the ligation.<br>
To check for self-closing vectors a vector-only sample will be ligated.
To check for self-closing vectors a vector-only sample will be ligated.
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<br><br>
==Materials==  
==Materials==  
*PCR tubes/PCR plate
*PCR tubes/PCR plate
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*T4 DNA ligase
*T4 DNA ligase
*T4 DNA ligase buffer
*T4 DNA ligase buffer
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<br>
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<i>Note: All materials should be held on ice during preparation.</i>
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Note: All materials should be held on ice during preparation.
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<br><br>
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==Protocol==
==Protocol==
{| border="1"
{| border="1"
!align="left"|ratio 1:1
!align="left"|ratio 1:1
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!align="right"|ul
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!align="right"|µl
|-
|-
|Vector
|Vector
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|- style="font-style:italic;"
|- style="font-style:italic;"
|Total
|Total
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|align="right"|10 ul
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|align="right"|10 µl
|}<br>
|}<br>
{| border="1"
{| border="1"
!align="left"|ratio 1:2
!align="left"|ratio 1:2
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!align="right"|ul
+
!align="right"|µl
|-
|-
|Vector
|Vector
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|- style="font-style:italic;"
|- style="font-style:italic;"
|Total
|Total
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|align="right"|10 ul
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|align="right"|10 µl
|}<br>
|}<br>
{| border="1"
{| border="1"
!align="left"|ratio 1:5
!align="left"|ratio 1:5
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!align="right"|ul
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!align="right"|µl
|-
|-
|Vector
|Vector
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|- style="font-style:italic;"
|- style="font-style:italic;"
|Total
|Total
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|align="right"|10 ul
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|align="right"|10 µl
|}<br>
|}<br>
{| border="1"
{| border="1"
!align="left"|Vector-only
!align="left"|Vector-only
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!align="right"|ul
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!align="right"|µl
|-
|-
|Vector
|Vector
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|- style="font-style:italic;"
|- style="font-style:italic;"
|Total
|Total
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|align="right"|10 ul
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|align="right"|10 µl
|}<br>
|}<br>
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1. Incubate for ON at 16C.<br>  
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1. Incubate ON at 16&deg;C.<br>  
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2. Incubate for 20min at 80C to heat kill.<br>
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2. Incubate for 20 min at 80&deg;C to heat kill.<br>
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3. Use 2ul of ligation to transform into competent cells.<br>
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3. Use 2 µl of ligationmix to transform into [https://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Transforming_Competent_Cells chemically competent cells] or [https://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Transforming_electrocompetent_Cells electro competent cells].<br>
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{{:Team:Amsterdam/Footer}}
{{:Team:Amsterdam/Footer}}

Latest revision as of 00:36, 22 September 2011

Ligation

After following our digestion protocol a ligation can be performed.
Different ratios of vector:insert are used, to get the best result out of the ligation.
To check for self-closing vectors a vector-only sample will be ligated.

Materials

  • PCR tubes/PCR plate
  • vector and insert DNA
  • dH20
  • T4 DNA ligase
  • T4 DNA ligase buffer

Note: All materials should be held on ice during preparation.

Protocol

ratio 1:1 µl
Vector 1
Insert 1
T4 DNA ligase buffer 1
Ligase 1
H2O 6
Total 10 µl

ratio 1:2 µl
Vector 1
Insert 2
T4 DNA ligase buffer 1
Ligase 1
H2O 5
Total 10 µl

ratio 1:5 µl
Vector 1
Insert 5
T4 DNA ligase buffer 1
Ligase 1
H2O 2
Total 10 µl

Vector-only µl
Vector 1
Insert 0
T4 DNA ligase buffer 1
Ligase 1
H2O 7
Total 10 µl

1. Incubate ON at 16°C.
2. Incubate for 20 min at 80°C to heat kill.
3. Use 2 µl of ligationmix to transform into chemically competent cells or electro competent cells.


Retrieved from "http://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Ligations"