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| ='''Ligation'''=
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| ''Time: 15 min''
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| ''Reference: NEB T4 ligase technical bulletin
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| http://www.neb.com/nebecomm/TechBulletinFiles/techbulletinM0202.pdf''
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|
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| ===<font color="red">Pre</font>===
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| *Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio.
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| ===<font color="green">Ligation</font color>===
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| #for 20 μL reaction volume, to eppendorf add:
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|
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| {| border="1" cellspacing="0" cellpadding="1" align="center"
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| ! Stuff
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| ! Volume (uL)
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| |-
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| |insert
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| |x
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| |-
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| |vector
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| |y
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| |-
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| |dH2O
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| |z
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| |-
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| |10X T4 ligase buffer
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| |2
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| |-
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| |T4 ligase
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| |0.5
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| |-
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| !Total
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| |20
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| |}
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| where x is usually 5-15 uL, y=[1,5] uL.
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| 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.
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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 1 hr. Proceed directly to transformation.
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| [http://openwetware.org/wiki/IGEM:PennState Main]
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