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Team:TUDelft/Protocols
From 2008.igem.org
[http://www.tudelft.nl ]
[http://www.tudelft.nl ]
Contents |
Protocols
Transformations
Standard transformation procedure
- Remove competent cells from -80, let thaw for 10 min on ice and aliquot in 50 ul amounts.
- add 2-5 ul of vector, usually in H2O, to 50 ul cells, no mixing by pipet due to shear induction.
- keep on ice for 20 minutes (vector spreading through volume)
- heat shock (42°C) for 45 seconds
- keep on ice for 2 minutes
- add 200 ul SOC, put on 37°C for 1 hour or longer with agitation.
- plate out 250 ul on appropriate antibiotics.
Restrictions
Try to do a restriction in a relatively large volume. As a rule of thumb, use a volume of 50 ul / 500 ng DNA.
- Calculate the amount of DNA you want to use
- add H2O
- add 10 x H buffer (Roche)
- add your calculated amount of DNA
- add 0.5 ul of each enzyme. Keep in mind 0.5 ul = 5 U, where 1 U is defined as the amount of enzyme cutting 1000 ng of DNA / hour, so for extremely large amounts of DNA adjust this.
- keep on 37°C for 2-3 hours.
Ligation
First make sure you have purified the DNA after restriction. Ligation should be in a small volume, so elute your DNA from the column in a small volume/high concentration.
- add H2O
- add 10 x ligation buffer
- add backbone and insert (theoretically in a 1:3 or 1:4 ratio, for 3A assembly it seemed to work at 1:1 ratios possibly better)
- add 1 ul of T4 Ligase.
