Engineering BioBrick vectors from BioBrick parts/DNA ligation: Difference between revisions
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(New page: ==Materials== *[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA ligase] from [http://www.neb.com/ NEB] *Deionized, sterile H<sub>2</sub>O *Purified, linearized destination ve...) |
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==Materials== | ==Materials== | ||
*[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA | *[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA Ligase] from [http://www.neb.com/ NEB] | ||
*10X T4 DNA Ligase buffer | |||
*Deionized, sterile H<sub>2</sub>O | *Deionized, sterile H<sub>2</sub>O | ||
*Purified, linearized destination vector (in H<sub>2</sub>O) | *Purified, linearized destination vector (in H<sub>2</sub>O) | ||
*Purified, linearized prefix part (in H<sub>2</sub>O) | *Purified, linearized prefix part (in H<sub>2</sub>O) | ||
*Purified, linearized suffix part (in H<sub>2</sub>O) | *Purified, linearized suffix part (in H<sub>2</sub>O) | ||
*Sterile 0.6mL plastic tubes | |||
==Ligation | ==Ligation mix== | ||
*2-4 μL each purified, linearized DNA | *2-4 μL each purified, linearized DNA | ||
* | *1X Ligase buffer | ||
*200 units T4 DNA Ligase | *200 units T4 DNA Ligase | ||
deionized H<sub>2</sub>O to 10μL | deionized H<sub>2</sub>O to 10μL | ||
| Line 16: | Line 18: | ||
==Procedure== | ==Procedure== | ||
#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube | Vortex all reagents before use. | ||
#Add | |||
#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube. | |||
#Add ligation buffer. <br>Vortex buffer before pipetting to ensure that it is well-mixed. <br>Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. It is recommended that you aliquot the Ligation Buffer into smaller quantities. | |||
#Add prefix part, suffix part and destination vector to the tube. | #Add prefix part, suffix part and destination vector to the tube. | ||
#Add T4 DNA | #Add T4 DNA Ligase. <br>Vortex ligase before pipetting to ensure that it is well-mixed. <br>Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add the correct amount, just touch your tip to the surface of the liquid when pipetting. | ||
#Incubate 20 minutes on the benchtop at room-temperature. | #Incubate 20 minutes on the benchtop at room-temperature. | ||
#Place on ice until transformation. | #Place on ice until transformation. | ||
[[Category:DNA]] [[Category:In vitro]] [[Category:Protocol]] | [[Category:DNA]] [[Category:In vitro]] [[Category:Protocol]] | ||
Latest revision as of 08:42, 18 March 2008
Materials
- T4 DNA Ligase from NEB
- 10X T4 DNA Ligase buffer
- Deionized, sterile H2O
- Purified, linearized destination vector (in H2O)
- Purified, linearized prefix part (in H2O)
- Purified, linearized suffix part (in H2O)
- Sterile 0.6mL plastic tubes
Ligation mix
- 2-4 μL each purified, linearized DNA
- 1X Ligase buffer
- 200 units T4 DNA Ligase
deionized H2O to 10μL
Procedure
Vortex all reagents before use.
- Add appropriate amount of deionized H2O to sterile 0.6 mL tube.
- Add ligation buffer.
Vortex buffer before pipetting to ensure that it is well-mixed.
Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. It is recommended that you aliquot the Ligation Buffer into smaller quantities. - Add prefix part, suffix part and destination vector to the tube.
- Add T4 DNA Ligase.
Vortex ligase before pipetting to ensure that it is well-mixed.
Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add the correct amount, just touch your tip to the surface of the liquid when pipetting. - Incubate 20 minutes on the benchtop at room-temperature.
- Place on ice until transformation.
