Ligation Troubleshooting SOP
Contents
1. Dialyze for 20 minutes if electroporating Ligation efficiency was marginally decreased by Ligation Troubleshooting SOP 2011 1 Doing a 1 hr ligation at room temperature 2 Using 100 ng vector 3 Using insert vector molar ratios of 5 1 and 1 1 Ligation efficiency was noticably decreased x100 by 1 Sticky end ligation with a larger insert 5 2 kb vector 2 6 kb insert 2 Blunt end ligation Ligation efficiency was severely decreased x10000 by 1 Using DNA fragments that have been exposed to UV during the gel extraction procedure can avoid by blind excision or by using a black light or 365nm UV transilluminator instead of the usual 312nm type 2 a Using the transformation without heat inactivation the ligation probably would ve been fine PEG promotes ligation but inhibits transformation For additional troublshooting check out the NEB FAQ page for T4 ligation 1 http www neb com nebecomm products faqproductM0202 asp 339 3 A1 Ligation failed because there was no ATP or Mg2 Use the supplied buffer or add ATP to a compatible buffer The ATP in buffers older than one year may have degraded enough to cause problems When supplementing with ATP be sure to use ribo ATP as deoxyribo ATP will not work Ligation failed due to high salt or EDTA in the reaction Clean up the DNA CIP BAP or SAP not completely inactivated from dephosphorylation step Follow the recommended procedure to remove the phosphatase Liga2. Ligation Troubleshooting SOP 2011 Ligations Troubleshooting even if am doing a gel extraction e After ligation would also do another digest with a single cut enzyme that will cut your vector not your insert and run on a gel to verify the size Run it next to vector alone cut with the same single cut enzyme and you should see the 500bp difference to make sure your insert is ligated properly Then you can proceed with the transformation In order to test your ligase try addingsome to DNA ladder in ligase buffer of course Treat at RT for 15 20 minutes then run on a gel If you don t have a change in the ladder everything sfited up your ligase is a the problem gt Usual ratio Vektor Insert 1 3 e your vector to insert N But by sure restrictase was right inactivated by temperature OR Do not use kit for purification but do it old way Ligation ratio should by 1 3 vector insert since ligation is bimoleculaar reaction http openwetware org wiki DNA Ligation 10uL Ligation Mix Larger ligation mixes are also commonly used 1 0 uL 10X T4 ligase buffer 6 1 molar ratio of insert to vector 10ng vector Add 8 5 vector and insert volume yl ddH20 0 5 uL T4 Ligase Achtung Ligase Puffer ist sehr instabil ATP k nnen bei l ngerem RT kaputt gehen 1 Remember that the buffer contains ATP so repeated freeze thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation 2
3. tion produced only linear DNA because the DNA concentration was too high Keep the total DNA concentration between 1 10 ug ml 4 Too much ligation mixture was added to the cells Add between 1 5 ul to 50 ul competent cells 5 The ligation mix contained PEG and was incubated overnight Extended ligation with PEG causes a drop off in transformation efficiency This could be due to the gradual production of large linear pieces of DNA that can inhibit transformation The buffer for the Quick Ligation Kit NEB M2200 contains PEG 6 The ligation mix was not purified prior to electroporation The buffer must be removed or a spark will be generated by the salt Dialyse the sample or use a spin column to purify The PEG in the Quick Ligation Kit Buffer NEB M2200 prevents sparking but it also prevents electroporation PEG must be removed using a spin column Q9 Can T4 DNA Ligase be heat inactivated A9 Yes heat at 65 C for 20 minutes Do not heat inactivate if there is PEG in thereaction buffer Quick Ligation Kit buffer NEB M2200 because transformation will be inhi b i t e d
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