Team:Newcastle/Ligation

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(Procedures)
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For sticky end ligations incubate on the bench of 3-4 hours or overnight.
For sticky end ligations incubate on the bench of 3-4 hours or overnight.
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Important point to note is that 1:3 dilution means that during ligation, for 1 unit of vector, there is 3 units of insert and for 1:5 ligation, for 1 unit of vector there is 5 units of insert.
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Important point to note is that 1:3 dilution means that during ligation, for 1 unit of vector, there are 3 units of insert and in 1:5 ligation, for 1 unit of vector there is 5 units of insert.
==Notes==
==Notes==

Revision as of 08:51, 30 July 2010

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Ligation

Procedures

Set up the ligation mix according:

Reagents 1:3(μl) 1:5(μl) Vector(μl)
Vector 1 1 1
Insert 3 5 N/A
10X BUFFER 1 1 1
T4 Ligase 1 1 1
H2O 4 2 7
Total Volume 10.0 10.0 10.0

For sticky end ligations incubate on the bench of 3-4 hours or overnight.

Important point to note is that 1:3 dilution means that during ligation, for 1 unit of vector, there are 3 units of insert and in 1:5 ligation, for 1 unit of vector there is 5 units of insert.

Notes

  • For standard ligation 100-200ng of vector DNA is required - can be checked using nanodrop
  • A ligation calcultion can be used however a 1:3 and a 1:5 ratio tends to be used instead.


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