Protocol 9 - Ligation of Restriction Enzyme Cut Genomic DNA into Restriction
Enzyme Cut Bluescript© Phagemid
Buffer
rATP
Frag DNA
Vector DNA
stock conc.
desired conc.
T4 ligase
ddH2O
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FINAL VOL.
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volume to
add
Supplies needed:
Equipment needed:
- 10X T4 DNA Ligase Buffer
- 10 mM rATP
- restriction enzyme cut DNA fragments
- restriction enzyme cut Bluescript© phagemid
- 1.5 ml sterile microcentrifuge tubes (1/student)
- ddH2O
- microcentrifuge
- P-10, 100 micropipettors
- rack to hold microfuge tubes
Important Notes to Remember:
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Use a different pipette tip for each component of the reaction so as to NOT
contaminate the stock solutions.
As usual, it is very important that you employ sterile technique to avoid
contamination.
Only remove the enzyme from the freezer or from the ice when you are prepared
to add it. When any enzyme is off the ice for a prolonged period, it can lose
activity over time if it is not on ice or in the freezer. Minimize the time it is
unfrozen.
If you are using genomic DNA, avoid excessive pipetting in order to avoid
sheering which may cause undesired breakage.
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1. Determine the final volume of the reaction you wish to perform (e.g. 50 μl).
Then, based on the stock concentrations, calculate the necessary amount of each
component of the reaction, calculating the amount of water needed last.
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2. Add these components to your 1.5 ml microcentrifuge reaction tube in the
following order:
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ddH2O
10 X T4 DNA Ligase Buffer
rATP's
Digested insert DNA fragments
Digested Bluescript© phagemids
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3. Mix these components by gently flicking the tube with your finger. Use the
mini-microcentrifuge to quickly spin the contents at about 3000 RPM for about 3
seconds.
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4. Add the T4 DNA ligase and incubate the reaction at room temperature for the
(22°C) appropriate for 2 to 24 hours.
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