Setting-up a LigationGene Jockeying: Tricks of the Trade so that your Ligations work Every Time!Bevin EngelwardWe will firstreview a simpleexample of a ligation.Know Your Vectors!-Find out as much as you can about your vector. Promoter Ori -Download sequence information from the web.-Create a restriction map on-line.-Keep very careful records of your design plans.Safety Checks:-Never trust any map if it is not commercially available. -Always check all sites that you plan to use before setting off to do any ligations.-Watch out for methylation sensitive sites.Picking Restriction Enzyme Sites for PCR Product Insert-They must not be in the insert-Each must be unique in the vector-Ideally they are compatible to be cut together in the same buffer-Remember to plan for a “kill” site (if using A and C, B is a kill site if it is unique to the vector an absent from the insert).AB CA cool way to add a killsite is to use compatiblecohesive ends, such as ApoI and EcoRIDesigning PCR Primers-Melting temp of the section that matches the template should be ~60oC-Make sure your sites are far enough from the ends! -Make sure your primers don’t anneal to each other or to themselves. -Check the newly created sequence for expected sites and unexpected sites.Preparing the Backbone for Ligation-When double digesting, set up three digests -preparative with both enzymes (about 1-5 ug)-under the same conditions, cut vector with each enzyme separately to make sure each cut completely-Be sure to run some uncut vector to see where the unwanted DNA is expected to be.Vector PreparationBamHI and XbaI alone show complete digestion.Uncut shows where the “invisible” DNA is that you want to avoid duringpurification.Typical Pitfalls in Preparing Vector-DNA is not completely digested.-DNA is overloaded on the preparative gel so that purified linearDNA is mixed with uncut DNA (even invisible amounts of uncut DNA can be a problem!).Setting up the PCR Reaction to Prepare Insert-Use “touchdown” to get good specificity and yield.-Be sure to use conditions optimal for your polymerase. -Use High Fidelity PCR to avoid unwanted mutations! Taq is highly prone to errors!-Cleaner templates yield better results.Preparing the PCR Product for Ligation-Use a PCR cleanup kit before digesting PCR products.http://www.millipore.com/publications.nsf/docs/PF1517EN00Tips on Getting Complete Digestion-Use the optimal buffer conditions. -Cut overnight at room temp or cut at 37oC in an incubator.-Always run uncut next to cut DNA to confirm complete digestion.-Set up parallel digests of control DNA.Check the quality of the PCRproducts after amplification(load 5 ul out of 100)Digest the PCR products in parallelwith control plasmid that you knowwill be linearized. Make sure control is completely cut before proceeding.Run your PCR product on a preparative gel by combining wells.Use a fresh blade.Cut the band out(quickly! Avoid UV!)Trim off excess gel.Gel purify using QiaexQiaex Purification Kit-Follow kit instructions.-Make sure everything you use is clean! (razor blade, petri dish etc.)-Minimize UV!!-Exception to Kit Instructions: At the last step, elute the DNA first into 10 ul of EB buffer, then redo the elution using 7 ul EB buffer.Always, without fail, run a pregel before your ligation:-run 1 ul and 1ul at 1:10 dilution of the gel purified insert and vector-run these samples on the same gel so that you can compareconcentrations directlyIn this example, the backbone is similar in concentration to 3d and f.Estimating the Concentration of Your DNA Before Ligation-Do not trust UV spec information, always look at the DNA on a gel. Even degraded DNA can give a decent spec readout.-Always run 1:10 dilutions of your DNA.-The minimum amount of DNA that you can see on a gel is about 3 ng.-Use a concentration standard.Setting Up the Ligation-Use no less than 25 ng of vector (this is the lower limit). Ideally, use about 100 ng vector.-Mix DNA at a 4:1 Insert:Vector molar ratio. -Try hard to keep it down to a 10 ul volume. Add ligase last. 1ul is a vast excess of ligase.-Do two control ligations, one with vector alone, one with insert alone.Insert vector standard (100 ng/ul)1 1:10 1 1:10 1 1:10From this gel, we estimate that the vector is 50 ng/ul and the insert is 10 ng/ul.Setting-up a LigationSize ConcentrationVector 5.8kb 50 ng/µlInsert 700bp 10ng/µlLigation should contain 100ng of vector.The vector is ~8X larger than the insert, so that equal ng shouldyield a 1:8 molar ratio of vector:insert.4X insert Control(no insert) Control(no vector)2µl vector (100ng) 2µl vector (100ng) 5µl insert(50ng) 5µl insert (50ng) 1µl 10X ligase buffer 1µl 10X buffer1µl 10X ligase buffer 0.5µl T4 DNA ligase 0.5µl DNA ligase0.5µl T4 DNA ligase 6.5 µl H20 3.5 µl H201.5 µl H20 10 µl 10 µl10 µl“Killing” the LigationTake advantage of “kill” sites whenever possible. -Ideally, destroy both parent vectors. Procedure:-Heat inactivate the ligase by heating to 65oC for 10 min.-Add 4 volumes of kill-enzyme in its buffer to the ligationreaction.-Incubate 37oC for 1-2 h.-It is recommended that you precipitate (or clean up and then precipitate) your ligation before popping into cells. Precipitation will remove salts and concentrate the DNA.It has been noted that ligase itself inhibits DNA from getting into competant cells.Getting the Ligation Into Cells-Most importantly, be certain that your competent cells are