USTC/Notebook/DNA ligation

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(Difference between revisions)
 
Line 20: Line 20:
*'''9µl''' DNA plasmids
*'''9µl''' DNA plasmids
*'''1µl''' biobricks
*'''1µl''' biobricks
-
*'''10ul''' T4 ligase
+
*'''10ul''' Solution I (T4 ligase and buffer)
*'''MIX''' the reaction by stiring  
*'''MIX''' the reaction by stiring  
*Incubate the reaction at 16*C for 2hrs or overnight to ensure complete ligation.
*Incubate the reaction at 16*C for 2hrs or overnight to ensure complete ligation.

Latest revision as of 11:56, 28 October 2008

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DNA Ligation

DNA ligases are used with restriction enzymes to insert genes into plasmids.


Materials

  • DNA plasmids double digested previously(eg.XbaI and PstI); . Measure concentration in Nanodrop beforehand.
  • Biobricks double digested previously(eg.SpeI and PstI )
  • equal amount of T4 ligase.


Procedure

The following volumes apply to a 20µl DNA ligation.

add the following components to PCR tube:

  • 9µl DNA plasmids
  • 1µl biobricks
  • 10ul Solution I (T4 ligase and buffer)
  • MIX the reaction by stiring
  • Incubate the reaction at 16*C for 2hrs or overnight to ensure complete ligation.