Difference between revisions of "Team:CCU Taiwan/Project/Protocol"
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<p class="description">1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins). </p> | <p class="description">1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins). </p> | ||
Revision as of 17:34, 21 July 2018
Transform (homemade e-coli dh5α)
1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).
2.Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
3.Mix 5 μl of DNA into 50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
4.Incubate the competent cell/DNA mixture on ice for 30 mins.
5.Heat shock each transformation tube into a 42°C water bath for 90 secs.
6.Put the tubes back on ice for 5 min.
7.Add 50 μl LB to the bacteria and grow in 37°C shaking incubator for 180 min.
8.Spin down
9.Plate all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic (Chloramphenicol).
10.Incubate plates at 37°C overnight.