"
Team:University College London/Protocols/T4dnaligase
From 2012.igem.org
(Difference between revisions)
(Created page with "== Transformation Protocol 2 == </noinclude> Nuclease positive cells {| class="bigtable" |- ! COMPONENT !! 20 μl REACTION) |- | 10X T4 DNA Ligase Buffer || 2 μl |- | Vector ...") |
Erinoerton (Talk | contribs) |
||
| (6 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
| - | + | 1. Set up the following reaction in a microcentrifuge tube on ice. | |
| - | + | (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert.) | |
| - | |||
{| class="bigtable" | {| class="bigtable" | ||
|- | |- | ||
| Line 16: | Line 15: | ||
|- | |- | ||
| T4 DNA Ligase || 1 μl | | T4 DNA Ligase || 1 μl | ||
| - | |- | + | |} |
| + | |||
| + | 2. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature. | ||
| + | |||
| + | 3. Gently mix the reaction by pipetting up and down and microfuge briefly. | ||
| + | |||
| + | 4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. | ||
| + | |||
| + | 5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation). | ||
| + | |||
| + | 6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. | ||
| + | |||
Latest revision as of 23:23, 26 September 2012
1. Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert.)
| COMPONENT | 20 μl REACTION) |
|---|---|
| 10X T4 DNA Ligase Buffer | 2 μl |
| Vector DNA (3 kb) | 50 ng (0.025 pmol) |
| Insert DNA (1 kb | 50 ng (0.076 pmol) |
| Nuclease-free water | to 20 μl |
| T4 DNA Ligase | 1 μl |
2. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
3. Gently mix the reaction by pipetting up and down and microfuge briefly.
4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
