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Fab library constructing-ligation/transformation problem

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Fab library constructing-ligation/transformation problem

Postby FABphage » Tue Jan 19, 2010 9:54 pm

I am working on constructing a phage library to express antibodies and currently I am doing the Fab library constructing work. I did PCR and got light chain (LC) products. There is a LC stuffer in the vector so I digested and gel purified the digested phagemid vector and the LC stuffer as a control. I tried to ligate the digested LC or LC stuffer with the vector. To test, I transformed the ligation product into DH5-alpha competent cells using regular heat shock transformation method. After picking colonies and digesting the miniprep products, I run a gel to check and the results are shown in the picture attached. From left to right, lanes 1-5 are the 5 colonies of vector-LC ligation while lanes 6-10 are the vector-LC stuffer colonies. Lane 13 is the purified vector as control. Apparently, there is some problem with the LC purification so the ligation-transformation did not work, I think? I did gel purify the LC product so I don’t know what to do right now. Any suggestion would be greatly appreciated!

011810 miniprep.jpg
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