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Cloning of large fragment

Cloning of large fragment - Molecular Biology Techniques

Cloning of large fragment - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 03-03-2012, 09:34 AM
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Default Cloning of large fragment



Good day.

i failed to clone a 2.2 kb gene in to a vector (about 12 kb). anybody can give me some oppinion and suggestion? below are the details

vector:
TAKARA pRI201-AN. cut with SacI, treated with T4 DNA polimerase to produce blunt ends, the cut with NdeI to product a linear vector with sticky end at one side and blunt end at another side. gel purified

insert:
PCR product amplified with Pfu dna polymerase ( blunt end), cut with NdeI at to produce stiky end at one side. ge purified

ligation:
90ng of vector and about 47ng of insert. (vector: insert =1:3)
in 20ul reaction

Transformation:
heat shock


Result:
all of the colonies i obtained are with out insert, seem like the vector ligate with another vector ( sticky end bind with sticky end, blunt end with blunt)

I really hpe somebody can help me to solve this problem.
Thank you very much.
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