Endy:DNA ligation using T4 DNA ligase: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Barry Canton (talk | contribs) |
Barry Canton (talk | contribs) No edit summary |
||
| Line 8: | Line 8: | ||
*Purified, linearized insert (likely in H<sub>2</sub>O or EB) | *Purified, linearized insert (likely in H<sub>2</sub>O or EB) | ||
==10μl Ligation Mix== | ==Procedure== | ||
===10μl Ligation Mix=== | |||
''Larger ligation mixes are also commonly used'' | ''Larger ligation mixes are also commonly used'' | ||
*1.0 μL 10X T4 ligase buffer | *1.0 μL 10X T4 ligase buffer | ||
| Line 14: | Line 15: | ||
*Add (8.5 - vector and insert volume)μl ddH<sub>2</sub>O | *Add (8.5 - vector and insert volume)μl ddH<sub>2</sub>O | ||
*0.5 μL T4 Ligase | *0.5 μL T4 Ligase | ||
===Calculating Insert Amount=== | |||
==Calculating Insert Amount== | |||
<math> {Insert\ Mass\ in\ ng} = 6\times\left[\frac{{Insert\ Length\ in\ bp}}{{Vector\ Length\ in\ bp}}\right]\times{Vector\ Mass\ in\ ng} </math> | <math> {Insert\ Mass\ in\ ng} = 6\times\left[\frac{{Insert\ Length\ in\ bp}}{{Vector\ Length\ in\ bp}}\right]\times{Vector\ Mass\ in\ ng} </math> | ||
===Method=== | |||
== | |||
#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube | #Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube | ||
#Add 1 μL ligation buffer to the tube. <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. | #Add 1 μL ligation buffer to the tube. <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. | ||
Revision as of 19:30, 12 July 2006
Materials
- T4 DNA Ligase
- 10x T4 DNA Ligase Buffer
- Deionized, sterile H2O
- Purified, linearized vector (likely in H2O or EB)
- Purified, linearized insert (likely in H2O or EB)
Procedure
10μl Ligation Mix
Larger ligation mixes are also commonly used
- 1.0 μL 10X T4 ligase buffer
- 6:1 Molar ratio of insert to vector (~10ng vector)
- Add (8.5 - vector and insert volume)μl ddH2O
- 0.5 μL T4 Ligase
Calculating Insert Amount
[math]\displaystyle{ {Insert\ Mass\ in\ ng} = 6\times\left[\frac{{Insert\ Length\ in\ bp}}{{Vector\ Length\ in\ bp}}\right]\times{Vector\ Mass\ in\ ng} }[/math]
Method
- Add appropriate amount of deionized H2O to sterile 0.6 mL tube
- Add 1 μL ligation buffer to the tube.
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. - Add appropriate amount of insert to the tube.
- Add appropriate amount of vector to the tube.
- Add 0.5 μL 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 only 1 μL, just touch your tip to the surface of the liquid when pipetting. - Let the 10 μL solution sit at 22.5°C for 30 mins
- Denature the ligase at 65°C for 10min
- Dialyze for 20 minutes if electroporating
- Use disks shiny side up
- Store at -20°C
Notes
Make sure the buffer is completely melted and dissolved. Precipitate is DTT. Probably best to aliquot this buffer into smaller portions, to reduce the freeze/thaw cycles. In general, make sure the buffer still smells strongly like "wet dog" (Checking if the DTT is still good.)
