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Team:Amsterdam/Notebook/Protocols/Ligations
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*T4 DNA ligase buffer | *T4 DNA ligase buffer | ||
<br> | <br> | ||
| - | Note: All materials should be held on ice during preparation. | + | <b>Note: All materials should be held on ice during preparation.</b> |
==Protocol== | ==Protocol== | ||
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{| border="1" | {| border="1" | ||
!align="left"|ratio 1:1 | !align="left"|ratio 1:1 | ||
| - | !align="right"| | + | !align="right"|µl |
|- | |- | ||
|Vector | |Vector | ||
| Line 37: | Line 37: | ||
|- style="font-style:italic;" | |- style="font-style:italic;" | ||
|Total | |Total | ||
| - | |align="right"|10 | + | |align="right"|10 µl |
|}<br> | |}<br> | ||
{| border="1" | {| border="1" | ||
!align="left"|ratio 1:2 | !align="left"|ratio 1:2 | ||
| - | !align="right"| | + | !align="right"|µl |
|- | |- | ||
|Vector | |Vector | ||
| Line 60: | Line 60: | ||
|- style="font-style:italic;" | |- style="font-style:italic;" | ||
|Total | |Total | ||
| - | |align="right"|10 | + | |align="right"|10 µl |
|}<br> | |}<br> | ||
{| border="1" | {| border="1" | ||
!align="left"|ratio 1:5 | !align="left"|ratio 1:5 | ||
| - | !align="right"| | + | !align="right"|µl |
|- | |- | ||
|Vector | |Vector | ||
| Line 83: | Line 83: | ||
|- style="font-style:italic;" | |- style="font-style:italic;" | ||
|Total | |Total | ||
| - | |align="right"|10 | + | |align="right"|10 µl |
|}<br> | |}<br> | ||
{| border="1" | {| border="1" | ||
!align="left"|Vector-only | !align="left"|Vector-only | ||
| - | !align="right"| | + | !align="right"|µl |
|- | |- | ||
|Vector | |Vector | ||
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|- style="font-style:italic;" | |- style="font-style:italic;" | ||
|Total | |Total | ||
| - | |align="right"|10 | + | |align="right"|10 µl |
|}<br> | |}<br> | ||
| - | 1. Incubate for ON at | + | 1. Incubate for ON at 16°C.<br> |
| - | 2. Incubate for | + | 2. Incubate for 20 min at 80C to heat kill.<br> |
3. Use 2ul of ligation to transform into competent cells.<br> | 3. Use 2ul of ligation to transform into competent cells.<br> | ||
Revision as of 13:24, 17 September 2011
Ligation
After following our digestion protocol a ligation can be performed.
Different ratios of vector:insert are used, to get the best result out of the ligation.
To check for self-closing vectors a vector-only sample will be ligated.
Materials
- PCR tubes/PCR plate
- vector and insert DNA
- dH20
- T4 DNA ligase
- T4 DNA ligase buffer
Note: All materials should be held on ice during preparation.
Protocol
| ratio 1:1 | µl |
|---|---|
| Vector | 1 |
| Insert | 1 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 6 |
| Total | 10 µl |
| ratio 1:2 | µl |
|---|---|
| Vector | 1 |
| Insert | 2 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 5 |
| Total | 10 µl |
| ratio 1:5 | µl |
|---|---|
| Vector | 1 |
| Insert | 5 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 2 |
| Total | 10 µl |
| Vector-only | µl |
|---|---|
| Vector | 1 |
| Insert | 0 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 7 |
| Total | 10 µl |
1. Incubate for ON at 16°C.
2. Incubate for 20 min at 80C to heat kill.
3. Use 2ul of ligation to transform into competent cells.
