problem in cloning 1

cloningproblem · #1 03-25-2012, 02:32 AM

Hi,
i can't have clone 1.8 Kb insert in 4.7Kb vector in ECOR I & Sma I site, i can get back round vector, even though dephosphorylate vector i can,t get any transformed colonies, site compactibility for the vector of pcr product are ok, can any one help

cloningproblem · #2 03-29-2012, 07:38 AM

Hi,
i can't have clone 1.8 Kb insert in 4.7Kb vector in ECOR I & Sma I site, i can get back round vector, even though dephosphorylate vector i can,t get any transformed colonies, site compactibility for the vector of pcr product are ok, can any one help

mmorgan · #3 03-31-2012, 09:47 PM

Quote:

Originally Posted by cloningproblem

Hi,
i can't have clone 1.8 Kb insert in 4.7Kb vector in ECOR I & Sma I site, i can get back round vector, even though dephosphorylate vector i can,t get any transformed colonies, site compactibility for the vector of pcr product are ok, can any one help

Try the cloning without dephosphorylating (it's not necessary if you're using 2 different enzymes).

Make sure to do a + and - insert control to ensure that you have digested your plasmid enough (this is usually the problem).

cloningproblem · #4 04-02-2012, 07:52 AM

Hi,
thanks, and i tried on your way but it did in't work, i hav also tried with EcoRI site, couldn't possible to get it, do u hav any suggestion, please...

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problem in cloning 1

problem in cloning 1 - Molecular Cloning Forum

problem in cloning 1 - 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.