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USTC/Notebook/DNA ligation
From 2008.igem.org
(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
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.
