# IGEM:PennState/2006/Ligation

(Difference between revisions)
 Revision as of 14:53, 22 May 2008 (view source)← Previous diff Current revision (15:39, 5 June 2008) (view source)m (→Pre) Line 2: Line 2: ===Pre=== ===Pre=== - *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. + ===Ligation=== ===Ligation=== #for 20 μL reaction volume, to eppendorf add: #for 20 μL reaction volume, to eppendorf add:

# 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)