Endy:DNA ligation using T4 DNA ligase: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Felix Moser (talk | contribs) No edit summary |
Felix Moser (talk | contribs) No edit summary |
||
Line 1: | Line 1: | ||
=Quick ligase ligation= | |||
==Materials== | |||
*2x Quick ligase buffer (in 40µl aliquots; these are 1-time use since freeze-thaw cycles degrade the ATP in the buffer). | |||
*ddH2O | |||
*Purified, linearized vector (likely in H<sub>2</sub>O or EB) | |||
*Purified, linearized insert (likely in H<sub>2</sub>O or EB)''Italic text'' | |||
==Procedure== | |||
===For 10µl reaction=== | |||
''Larger volumes can be scaled up if needed'' | |||
*5 μL 2X Quick ligase buffer | |||
*6:1 Molar ratio of insert to vector (~10ng vector). Try to keep total DNA concentration <100ng/rxn for optimal efficiency. | |||
*Add (4.5 - vector and insert volume)μl ddH<sub>2</sub>O | |||
===Method=== | |||
#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 appropriate amount of insert to the tube. | |||
#Add appropriate amount of vector to the tube. | |||
#Add 0.5 μL 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 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 | |||
=T4 ligase ligation= | |||
==Materials== | ==Materials== | ||
*[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA Ligase] | *[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA Ligase] | ||
*10x T4 DNA Ligase Buffer | *10x T4 DNA Ligase Buffer | ||
*Deionized, sterile H<sub>2</sub>O | *Deionized, sterile H<sub>2</sub>O | ||
*Purified, linearized vector (likely in H<sub>2</sub>O or EB) | *Purified, linearized vector (likely in H<sub>2</sub>O or EB) | ||
*Purified, linearized insert (likely in H<sub>2</sub>O or EB) | *Purified, linearized insert (likely in H<sub>2</sub>O or EB)''Italic text'' | ||
==Procedure== | ==Procedure== | ||
===10μl Ligation Mix=== | ===10μl Ligation Mix=== | ||
Line 24: | Line 46: | ||
#Let the 10 μL solution sit at 22.5°C for 30 mins | #Let the 10 μL solution sit at 22.5°C for 30 mins | ||
#Denature the ligase at 65°C for 10min | #Denature the ligase at 65°C for 10min | ||
#Store at -20°C | #Store at -20°C | ||
==Notes== | ==Notes== | ||
Make sure the buffer is completely melted and dissolved. Precipitate is DTT (or BSA?). 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.) | Make sure the buffer is completely melted and dissolved. Precipitate is DTT (or BSA?). 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.) |
Revision as of 09:04, 24 June 2008
Quick ligase ligation
Materials
- 2x Quick ligase buffer (in 40µl aliquots; these are 1-time use since freeze-thaw cycles degrade the ATP in the buffer).
- ddH2O
- Purified, linearized vector (likely in H2O or EB)
- Purified, linearized insert (likely in H2O or EB)Italic text
Procedure
For 10µl reaction
Larger volumes can be scaled up if needed
- 5 μL 2X Quick ligase buffer
- 6:1 Molar ratio of insert to vector (~10ng vector). Try to keep total DNA concentration <100ng/rxn for optimal efficiency.
- Add (4.5 - vector and insert volume)μl ddH2O
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
T4 ligase ligation
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)Italic text
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
- Store at -20°C
Notes
Make sure the buffer is completely melted and dissolved. Precipitate is DTT (or BSA?). 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.)