IGEM:PennState/2006/Ligation

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m (<font color="green">Ligation</font color>: changed link format from external to internal)
Current revision (14:39, 5 June 2008) (view source)
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='''Ligation'''=
='''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''
 
===<font color="red">Pre</font>===  
===<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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*Estimate DNA concentration of restricted fragments.   Aim for a 3:1 insert:vector molar ratio (660 g/mole/base pair).
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** Where x is the desired concentration of insert, y is the desired concentration of vector, and n is the current ratio of their concentrations, a 3:1 ratio implies the relationship 3/n*y = x .
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** Additionally, the volumes of the substances is governed by the equation total volume = volX + volY + buffer. Usually the total volume we use for ligations is 40μL.
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===<font color="green">Ligation</font color>===
===<font color="green">Ligation</font color>===
#for 20 μL reaction volume, to eppendorf add:
#for 20 μL reaction volume, to eppendorf add:
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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.   
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 30 min.  Proceed directly to transformation.
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*For overnight ligation, store at 16C (15 hr)
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[[Penn_State_University_2006:Cloning|Cloning]]
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[[IGEM:PennState Main]]
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Current revision

Ligation

Pre

  • Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio (660 g/mole/base pair).
    • Where x is the desired concentration of insert, y is the desired concentration of vector, and n is the current ratio of their concentrations, a 3:1 ratio implies the relationship 3/n*y = x .
    • Additionally, the volumes of the substances is governed by the equation total volume = volX + volY + buffer. Usually the total volume we use for ligations is 40μL.

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.

  • For overnight ligation, store at 16C (15 hr)
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