Knight:DNA ligation using NEB Quick Ligation Kit

(Difference between revisions)
Jump to: navigation, search
 Revision as of 23:14, 20 June 2005 (view source)← Previous diff Current revision (13:22, 21 December 2007) (view source) (3 intermediate revisions not shown.) Line 1: Line 1: - ==Knight lab protocol== + ==Materials== - + - ===Materials=== + *We use the [http://www.neb.com/nebecomm/products/productM2200.asp Quick Ligation Kit] from [http://www.neb.com/ NEB] *We use the [http://www.neb.com/nebecomm/products/productM2200.asp Quick Ligation Kit] from [http://www.neb.com/ NEB] Line 8: Line 6: *Purified, linearized insert (in H2O) *Purified, linearized insert (in H2O) - ===Ligation Mix=== + ==Ligation Mix== This is the same [http://www.neb.com/nebecomm/products/protocol2.asp protocol] that NEB recommends. This is the same [http://www.neb.com/nebecomm/products/protocol2.asp protocol] that NEB recommends. Line 18: Line 16: *1 μL Quick Ligase *1 μL Quick Ligase - ===Calculating Insert Amount=== + ==Calculating Insert Amount== $\rm{Insert\ Mass\ in\ ng} = 3\times\left[\frac{\rm{Insert\ Length\ in\ bp}}{\rm{Vector\ Length\ in\ bp}}\right]\times \rm{Vector\ Mass\ in\ ng}$ $\rm{Insert\ Mass\ in\ ng} = 3\times\left[\frac{\rm{Insert\ Length\ in\ bp}}{\rm{Vector\ Length\ in\ bp}}\right]\times \rm{Vector\ Mass\ in\ ng}$ - ===Procedure=== + ==Procedure== #Add appropriate amount of deionized H2O to sterile 0.6 mL tube #Add appropriate amount of deionized H2O to sterile 0.6 mL tube Line 32: Line 30: #Place on ice until transformation. #Place on ice until transformation. #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. #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. - #''Optional''  Heat-inactivate by incubating at 65°C for 20 mins.  Then do a [[QIAquick PCR purification | purification]] step to remove PEG. (See notes below). + #''Optional''  Heat-inactivate by incubating at 65°C for 20 mins.  Then do a [[Purification of DNA | purification]] step to remove PEG. (See notes on [[DNA Ligation|DNA ligation]]. - + - ==Notes== + - #If you are having trouble with your ligation, there is an [http://www.neb.com/nebecomm/products/faqproductM2200.asp FAQ] to help. + [[Category:DNA]] [[Category:In vitro]] [[Category:Protocol]] - #Prior to the ligation, some heat their DNA slightly (maybe ~37°C) to melt any sticky ends which may have annealed improperly at low temperatures. + - #Tom has read that ligase can inhibit transformation.  By heat-inactivating the ligase, this inhibition can be avoided.  However, according to the NEB FAQ, heat-inactivation of PEG (which is present in the ligation reaction) also inhibits transformation, therefore a spin-column purification is recommended prior to transformation. + - #Tom learned that treating PCR products with proteinase K prior to restriction digest dramatically improves the efficiency of subsequent ligation reactions.  See Crowe JS, Cooper HJ, Smith MA, Sims MJ, Parker D, Gewert D. Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion. ''Nucleic Acids Res''. 1991 Jan 11;19(1):184. +

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

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

Procedure

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.
9. Optional Heat-inactivate by incubating at 65°C for 20 mins. Then do a purification step to remove PEG. (See notes on DNA ligation.