DNA ligation: Difference between revisions

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(Added Knight lab protocol. Some editing changes to Endy lab protocol.)
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'''Ligation Mix'''
=Endy lab protocol=
*Example - 10ul mix


*1.0 <math>\mu</math>L 10X T4 ligase buffer
==Ligation Mix==


*0.5 <math>\mu</math>L T4 Ligase
''Example - 10ul mix''


*6:1 Molar ratio of insert to vector (<math>\sim</math>10ng vector)
*1.0 &mu;L 10X T4 ligase buffer
*X<math>\mu</math>L dd<math>\rm{H_2O}</math> to bring the total to 10<math>\mu</math>L
*0.5 &mu;L T4 Ligase
*6:1 Molar ratio of insert to vector (~10ng vector)
*X &mu;L ddH<sub>2</sub>O to bring the total to 10&mu;L


==Calculating Insert and Vector Amounts==


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


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


 
*Let the 10 &mu;L solution sit at 22.5&deg;C for 30 mins
'''Reaction Conditions'''
*Denature the ligase at 65&deg;C for 10min
*Let the 10 <math>\mu</math>L solution sit at <math>22.5^o</math>C for 30min
*Denature the ligase at <math>65^o</math>C for 10min
*Dialyze for 20 minutes if electroporating
*Dialyze for 20 minutes if electroporating
*Use disks shiny side up
*Use disks shiny side up
*Store at -20C
*Store at -20&deg;C
 
=Knight lab protocol=
 
==Materials==
 
*We use the [http://www.neb.com/nebecomm/products/productM2200.asp Quick Ligation Kit] from [http://www.neb.com/ NEB]
*Deionized, sterile H<sub>2</sub>O
*Purified, linearized vector (in H<sub>2</sub>O)
*Purified, linearized insert (in H<sub>2</sub>O)
 
==Ligation Mix==
 
This is the same [http://www.neb.com/nebecomm/products/protocol2.asp protocol] that NEB recommends.
 
*X &mu;L vector (equivalent to 50 ng)
*Y &mu;L insert (3-fold molar excess, see below)
*10 &mu;L 2X Ligase Buffer
*(10 - X - Y) &mu;L deionized H<sub>2</sub>O
*1 &mu;L Quick Ligase
 
==Calculating Insert Amount==
 
<math> \rm{Insert\ Mass\ in\ ng} = 3\times\left[\frac{\rm{Insert\ Length\ in\ bp}}{\rm{Vector\ Length\ in\ bp}}\right]\times \rm{Vector\ Mass\ in\ ng} </math>
 
==Procedure==
 
If you are having trouble with your ligation, there is an [http://www.neb.com/nebecomm/products/faqproductM2200.asp FAQ] to help.
 
#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube
#Add 10 &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.  It is recommended that you aliquot the Ligation Buffer into smaller quantities.
#Add appropriate amount of insert to the tube.
#Add appropriate amount of vector to the tube.
#Add 1 &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.
#Incubate 5 mins on the benchtop.
#Place on ice until transformation.
#Generally 1 &mu;L of ligation mix is sufficient for either chemical transformation or electroporation.  The amount of salt in 1 &mu;L ligation mix should not cause arcing.

Revision as of 08:46, 12 May 2005

Endy lab protocol

Ligation Mix

Example - 10ul mix

  • 1.0 μL 10X T4 ligase buffer
  • 0.5 μL T4 Ligase
  • 6:1 Molar ratio of insert to vector (~10ng vector)
  • X μL ddH2O to bring the total to 10μL

Calculating Insert and Vector Amounts

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

Procedure

  • 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

Knight lab protocol

Materials

  • We use the Quick Ligation Kit from NEB
  • Deionized, sterile H2O
  • Purified, linearized vector (in H2O)
  • Purified, linearized insert (in H2O)

Ligation Mix

This is the same protocol that NEB recommends.

  • X μL vector (equivalent to 50 ng)
  • Y μL insert (3-fold molar excess, see below)
  • 10 μL 2X Ligase Buffer
  • (10 - X - Y) μL deionized H2O
  • 1 μL Quick Ligase

Calculating Insert Amount

[math]\displaystyle{ \rm{Insert\ Mass\ in\ ng} = 3\times\left[\frac{\rm{Insert\ Length\ in\ bp}}{\rm{Vector\ Length\ in\ bp}}\right]\times \rm{Vector\ Mass\ in\ ng} }[/math]

Procedure

If you are having trouble with your ligation, there is an FAQ to help.

  1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
  2. Add 10 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. It is recommended that you aliquot the Ligation Buffer into smaller quantities.
  3. Add appropriate amount of insert to the tube.
  4. Add appropriate amount of vector to the tube.
  5. Add 1 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. Incubate 5 mins on the benchtop.
  7. Place on ice until transformation.
  8. Generally 1 μL of ligation mix is sufficient for either chemical transformation or electroporation. The amount of salt in 1 μL ligation mix should not cause arcing.
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