From 2012.igem.org
(Difference between revisions)
|
|
| Line 1: |
Line 1: |
| - | [[Category:Material and Methods]]
| |
| - | === Restriction ===
| |
| | | | |
| - |
| |
| - |
| |
| - | === Ligation ===
| |
| - | # Combination of 50 ng of vector with a 2-fold molar excess of insert.
| |
| - | # Addition of an appropriate volume of T4 Buffer
| |
| - | # Addition of 0.5-1 µl T4 ligase
| |
| - | # Adjustment of volume to 20 µl with ddH2O
| |
| - | # Incubation over night at 16 °C
| |
| - | # Deactivation of ligase for 10 min at 65 °C
| |
| - |
| |
| - | === Chemical transformation ===
| |
| - | # Thawing of competent cells on ice
| |
| - | # Addition of 1 µL DNA
| |
| - | # Incubation on ice for 20 min
| |
| - | # Heat shock for 1 min at 42 °C
| |
| - | # Incubation on ice for 5 min
| |
| - | # Addition of 270 µL LB
| |
| - | # Incubation in thermoblock for 45 min (incubation time dependent on used vector!) at 37 °C and 300 rpm
| |
| - | # Smearing of cells on plates and incubation at 37°C (centrifuge it, discard supernatant, resuspend cells in rest-medium, 20 µL on plate)
| |
Latest revision as of 09:20, 1 August 2012
"
