Team:StanfordBrownSpelman/Lab Techniques5

Ligation & Chemically
Competent Transformation

Bioengineering Procedures

Ligation
(adapted from openwetware ligation protocol):

10 μl Recipe:
30-50 ng vector DNA (A calculator to make life easy)
1μl (10%) 10X T4 DNA ligase buffer
0.5μl (.5%) T4 DNA ligase
Top up w/ qH20 up to 10uL

Procedure
Usually heat inactivation of digests is sufficient; difficult ligations might require a proper cleanup
As often as possible, use isolated inserts and vectors to avoid unwanted ligations
If the reaction needs to be greater than 10μl, adjust amount of 10X ligase buffer and T4 DNA ligase so that they remain at 1% and .5% by volume, respectively
For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
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).

Chemically Competent Transformation

Materials:
1 aliquot of competent cells
2-4μl ligation mixture
500μl SOC media

Procedure
Thaw cells at 4°C for 5 minutes
Gently mix in ligation product
Incubate at 4°C for 20 min
Meanwhile, warm SOC media to 37°C
Heat shock at 42°C for 30 sec (45 sec for NEB5-alpha cells)
Return to 4°C for 1 min
Add 500μl pre-heated SOC
Incubate at 37°C for 1hr with shaking
Meanwhile, pre-heat plates to 37°C
Plate, one plate w/ 100μl, one plate w/ 150μl

      

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