IGEM:PennState/2006/Ligation

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2. For overnight ligation, add ice to cold water in styrofoam container to bring temp to 12-14ºC.  Incubate overnight (15 hr.).  Alternatively, for quicker ligation, ligate at RT for 30 min.  Proceed directly to transformation.
2. For overnight ligation, add ice to cold water in styrofoam container to bring temp to 12-14ºC.  Incubate overnight (15 hr.).  Alternatively, for quicker ligation, ligate at RT for 30 min.  Proceed directly to transformation.
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[http://openwetware.org/wiki/Penn_State_University_2006:Cloning Cloning]
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[[Penn_State_University_2006:Cloning|Cloning]]
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[http://openwetware.org/wiki/IGEM:PennState Main]
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[[IGEM:PennState Main]]

Revision as of 09:23, 22 May 2008

Ligation

Time: 15 min

Reference: NEB T4 ligase technical bulletin http://www.neb.com/nebecomm/TechBulletinFiles/techbulletinM0202.pdf

Pre

  • Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio.

Ligation

  1. for 20 μL reaction volume, to eppendorf add:
Stuff Volume (uL)
insert x
vector y
dH2O z
10X T4 ligase buffer 2
T4 ligase 0.5
Total 20

where x is usually 5-15 uL, y=[1,5] uL.

Also, always remember to do a negative control, which will be like the reaction above, but w/ no insert. Your transformation plates will then be an measure of any vector re-ligation/uncut background.

2. For overnight ligation, add ice to cold water in styrofoam container to bring temp to 12-14ºC. Incubate overnight (15 hr.). Alternatively, for quicker ligation, ligate at RT for 30 min. Proceed directly to transformation.

Cloning

IGEM:PennState Main

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