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USTC/Notebook/DNA ligation
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== DNA Ligation == | == DNA Ligation == | ||
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| + | 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. | ||
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.
