Endy:DNA ligation using T4 DNA ligase: Difference between revisions

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{{EndyLab}}
{{EndyLab}}
===Ligation Mix===
==Materials==


''Example - 10ul mix''
*[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA Ligase]
*10x T4 DNA Ligase Buffer
*Deionized, sterile H<sub>2</sub>O
*Purified, linearized vector (likely in H<sub>2</sub>O or EB)
*Purified, linearized insert (likely in H<sub>2</sub>O or EB)


==10&mu;l Ligation Mix==
''Larger ligation mixes are also commonly used''
*1.0 &mu;L 10X T4 ligase buffer
*1.0 &mu;L 10X T4 ligase buffer
**Make sure this 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.)
*6:1 Molar ratio of insert to vector (~10ng vector)
*Add (8.5 - vector and insert volume)&mu;l ddH<sub>2</sub>O
*0.5 &mu;L T4 Ligase
*0.5 &mu;L T4 Ligase
*6:1 Molar ratio of insert to vector (~10ng vector)


*Dilute with ddH<sub>2</sub>O to bring the total volume to 10&mu;L
==Calculating Insert Amount==


===Calculating Insert and Vector Amounts===
<math> \rm{Insert\ Mass\ in\ ng} = 6\times\left[\frac{\rm{Insert\ Length\ in\ bp}}{\rm{Vector\ Length\ in\ bp}}\right]\times \rm{Vector\ Mass\ in\ ng} </math>


<math> \rm{Insert\ Mass} = 6\times\left[\frac{\rm{Insert\ Length}}{\rm{Vector\ Length}}\right]\times \rm{Vector\ Mass} </math>
==Procedure==


===Procedure===
#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube
#Add 1 &mu;L 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 &mu;L 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 &mu;L, just touch your tip to the surface of the liquid when pipetting.
#Let the 10 &mu;L solution sit at 22.5&deg;C for 30 mins
#Denature the ligase at 65&deg;C for 10min
#Dialyze for 20 minutes if electroporating
#Use disks shiny side up
#Store at -20&deg;C


*Let the 10 &mu;L solution sit at 22.5&deg;C for 30 mins
==Notes==
*Denature the ligase at 65&deg;C for 10min
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.)
*Dialyze for 20 minutes if electroporating
*Use disks shiny side up
*Store at -20&deg;C

Revision as of 10:19, 12 January 2006

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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)

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{ \rm{Insert\ Mass\ in\ ng} = 6\times\left[\frac{\rm{Insert\ Length\ in\ bp}}{\rm{Vector\ Length\ in\ bp}}\right]\times \rm{Vector\ Mass\ in\ ng} }[/math]

Procedure

  1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
  2. 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.
  3. Add appropriate amount of insert to the tube.
  4. Add appropriate amount of vector to the tube.
  5. 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.
  6. Let the 10 μL solution sit at 22.5°C for 30 mins
  7. Denature the ligase at 65°C for 10min
  8. Dialyze for 20 minutes if electroporating
  9. Use disks shiny side up
  10. 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.)

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