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pLentiTOPO TA Cloning - Insert consistently in reverese direction when grown in Ecoli

pLentiTOPO TA Cloning - Insert consistently in reverese direction when grown in Ecoli - Molecular Cloning Forum

pLentiTOPO TA Cloning - Insert consistently in reverese direction when grown in Ecoli - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 07-26-2011, 01:25 AM
Pipette Filler
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Default pLentiTOPO TA Cloning - Insert consistently in reverese direction when grown in Ecoli



I'm hoping to get advice from some more experienced molecular biologists out there...


I am attempting to create an immortal cell line expressing a surface marker, but i have hit a road block. I am using a pLentiTOPO6.3 TA Cloning kit from Invitrogen. My ligations of my PCR insert into the pLentiTOPO vector ALWAYS result in vector/insert constructs with the insert in the wrong direction / orientation. The constructs are grown in Stbl3 E.coli, but I cannot understand why none of my colonies result in a vector/insert construct with the desired orientation of the insert.

My full length insert is 3.7Kb; truncated insert is 3.3kb. I have screened by either PCR, sequencing, or restriction digest about 100 colonies from my several ligation attempts. I have successfully isolated ONLY ONE truncated construct, but NOT ONE full-length construct in the correct orientation.

Your suggestions and/or advice is greatly appreciated :-)
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cloning , consistently , direction , ecoli , grown , insert , plentitopo , reverese


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