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Please help Cloning issues with smaI and pUNK1 vector - (Mar/24/2009 )

I am having problems cloning my promoter from the gene i am investigating into pUNK1 plasmid containing a lacz reporter gene. For my ligation 20 Ál of ligation mix was prepared using a 5:1 insert to vector ratio. The insert had been previously purified. and the vector had been vut with smaI. It contained 1 component of ligase buffer, 1 component of T4 ligase and 2 components of sterile distilled water. The mixture was flicked gently and spun for a few seconds in a desk top centrifuge at 14,000 rpm. The mixture was stored at 16˚C in a bucket of cold water for 18 hours before being placed in a water bath at 65˚C for 20 minutes to inactivate ligase activity. 14 Ál of sterile distilled water 4 Ál of buffer L and 2 Ál of XmaC1 a restriction enzyme were then added to the mixture before it was incubated at 37˚C for 1 hour. After doing this i transformed my vactor into DH5α. Now i have screened about 100 colonies through colony pcr and only 1 came back positive (all the rest had empty vectors). Then sequence analysis revelas the insert is in the wrong way round! other people in the labs have been having the same issue. Can anyone help me come up with an idea how to make this work?

-micro1510-

Can you clone in your promoter using something other than a blunt end ligation?

It is not clear, but did you dephosphorylate your vector?

-perneseblue-

try to increase ligase 2 to 5 times and incubate at 4C for more than 24 hours. That should give more +ve colonies

-Nrelo-