Team:Amsterdam/Notebook/Protocols/Ligations

From 2011.igem.org

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(Created page with "{{:Team:Amsterdam/Header}} =Ligations== After following our restriction digest protocol (which uses 500ng of DNA) you may follow these steps for ligation. 1. Add 11ul of dH20 2...")
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{{:Team:Amsterdam/Header}}
{{:Team:Amsterdam/Header}}
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=Ligations==
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=Ligation=
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After following our restriction digest protocol (which uses 500ng of DNA) you may follow these steps for ligation.
+
After following our 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>
 +
To check for self-closing vectors a vector-only sample will be ligated.
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1. Add 11ul of dH20
+
==Materials==
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2. Add 2ul from each sample you will be ligating (destination plasmid, and part)<br>
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*PCR tubes/PCR plate
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3. Add 2ul of T4 DNA Ligase Reaction Buffer<br>
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*vector and insert DNA
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4. Add 1ul of T4 DNA Ligase<br>
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*dH20
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5. Mix well, and spin down.<br>
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*T4 DNA ligase
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6. Incubate for 30min at 16C and 20min at 80C to heat kill.
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*T4 DNA ligase buffer
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7. Use 2ul of ligation to transform into competent cells.
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<br>
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Note: All materials should be held on ice during preparation.
 +
 
 +
==Protocol==
 +
 
 +
{| border="1"
 +
!align="left"|ratio 1:1
 +
!align="right"|ul
 +
|-
 +
|Vector
 +
|align="right"|1
 +
|-
 +
|Insert
 +
|align="right"|1
 +
|-
 +
|T4 DNA ligase buffer
 +
|align="right"|1
 +
|-
 +
|Ligase
 +
|align="right"|1
 +
|-
 +
|H2O
 +
|align="right"|6
 +
|- style="font-style:italic;"
 +
|Total
 +
|align="right"|10 ul
 +
|}<br>
 +
 
 +
{| border="1"
 +
!align="left"|ratio 1:2
 +
!align="right"|ul
 +
|-
 +
|Vector
 +
|align="right"|1
 +
|-
 +
|Insert
 +
|align="right"|2
 +
|-
 +
|T4 DNA ligase buffer
 +
|align="right"|1
 +
|-
 +
|Ligase
 +
|align="right"|1
 +
|-
 +
|H2O
 +
|align="right"|5
 +
|- style="font-style:italic;"
 +
|Total
 +
|align="right"|10 ul
 +
|}<br>
 +
 
 +
{| border="1"
 +
!align="left"|ratio 1:5
 +
!align="right"|ul
 +
|-
 +
|Vector
 +
|align="right"|1
 +
|-
 +
|Insert
 +
|align="right"|5
 +
|-
 +
|T4 DNA ligase buffer
 +
|align="right"|1
 +
|-
 +
|Ligase
 +
|align="right"|1
 +
|-
 +
|H2O
 +
|align="right"|2
 +
|- style="font-style:italic;"
 +
|Total
 +
|align="right"|10 ul
 +
|}<br>
 +
 
 +
{| border="1"
 +
!align="left"|Vector-only
 +
!align="right"|ul
 +
|-
 +
|Vector
 +
|align="right"|1
 +
|-
 +
|Insert
 +
|align="right"|0
 +
|-
 +
|T4 DNA ligase buffer
 +
|align="right"|1
 +
|-
 +
|Ligase
 +
|align="right"|1
 +
|-
 +
|H2O
 +
|align="right"|7
 +
|- style="font-style:italic;"
 +
|Total
 +
|align="right"|10 ul
 +
|}<br>
 +
 
 +
1. Incubate for ON at 16C.<br>
 +
2. Incubate for 20min at 80C to heat kill.<br>
 +
3. Use 2ul of ligation to transform into competent cells.<br>
{{:Team:Amsterdam/Footer}}
{{:Team:Amsterdam/Footer}}

Revision as of 13:57, 13 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 ul
Vector 1
Insert 1
T4 DNA ligase buffer 1
Ligase 1
H2O 6
Total 10 ul

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

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

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

1. Incubate for ON at 16C.
2. Incubate for 20min at 80C to heat kill.
3. Use 2ul of ligation to transform into competent cells.

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