IGEM:PennState/2006/Ligation: Difference between revisions
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='''Ligation'''= | ='''Ligation'''= | ||
===<font color="red">Pre</font>=== | ===<font color="red">Pre</font>=== | ||
*Estimate DNA concentration of restricted fragments. | *Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio (660 g/mole/base pair). | ||
===<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: | ||
| Line 37: | Line 33: | ||
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. | ||
*For overnight ligation, store at 16C (15 hr) | |||
Revision as of 11:53, 22 May 2008
Ligation
Pre
- Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio (660 g/mole/base pair).
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)
