IGEM:PennState/2006/Ligation: Difference between revisions
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===<font color="red">Pre</font>=== | ===<font color="red">Pre</font>=== | ||
*Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio (660 g/mole/base pair). | *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. | |||
===<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: | ||
Latest revision as of 12:39, 5 June 2008
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
- 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)
