ligation - (Aug/20/2007 )
hi all
i am trying to ligate a 600 base pairs gene with PET 28c vector
i have digested both vector and gene with BamH1 and Nde1
i have tried in all possibilities to ligate them
but after transformation i am not getting any colonies
i am sure that comp cells are ok i have checked
and ligase ia laso ok it is also working fine
please help me
thank you
NdeI is known as a fussy enzyme. Make sure it is cutting. It needs many bases of overhang if it is a linear fragment being cut.
hi
thank you for reply
i have checked out after digesting with Nde1
it is giving a linearised band in expected region
please help me what is the reason for not getting colonies
thank you
there are many many reasons why you may not have colonies. To list them all will be exhautive, (further compounded because there are many things considered standard procedure in one lab, but radical and strange in another. Even the condition and maintainence / hence accuracy of the gilson pipette.)
So, could you please explain in painful detail what you have done, Where does your insert come from. Where does your vector come from. What you have exactly done. How long do you digest your DNA for? Do you dephosphorylated your vector? How long for? How much DNA was dephosphorylated. Your ligation conditions, how many mols of vector and insert. Ligation conditions. Is your ligase and ligase buffer new. Has anybody else experienced problems with ligation?
How do you transform your cells?How much DNA do you transform? How long do you recover them from? How much antibiotics do you use?
Have you done any controls? What are the results? When you say no colonies do you mean no colonies at all, or no colonies with your plasmid?
So, could you please explain in painful detail what you have done, Where does your insert come from. Where does your vector come from. What you have exactly done. How long do you digest your DNA for? Do you dephosphorylated your vector? How long for? How much DNA was dephosphorylated. Your ligation conditions, how many mols of vector and insert. Ligation conditions. Is your ligase and ligase buffer new. Has anybody else experienced problems with ligation?
How do you transform your cells?How much DNA do you transform? How long do you recover them from? How much antibiotics do you use?
Have you done any controls? What are the results? When you say no colonies do you mean no colonies at all, or no colonies with your plasmid?
hi
i am amplifying my insert from a recominant plasmid with primers ,my vector from our lab only ,that i am isolating from cells and using for cloning ,
i have been digesting vector and insert for three and half hours ,being double digestion with BamH1 and Nde1 i thought no need to dephosporylate the vector ,so i didn't dephosporylated
my ligattion coditions i have tried at 16 , 22 , 4 ,degrees all of three for overnight some times with rapid ligation kit also for 3hrs
thae molar ratio i am not that much particular because i am not quantifying absorbance at 260
just by observing band in gel i used to go previous but after that
just assuming the minimum visible band intensity on agarose as 15-20 ng accordingly caliculated molar ratios and went for ligation from ratio 1:2 to 1:10
but i am not well aware of molar ratios and how to go with respect to that
previous and all i used to take 2 microlitre of plasmid approx (100ng ) and 8 micrlitre of insert and ligate a total of 20 microlitre of reaction mixture
incubation at 4 degree or 22 degree usually that time i used to get good colonies but with out my insert
i kept controls for ligase action and transformation efficiency in both i have got positive result
i have ligated single digested vector alone for checking action of ligase in that i have got colonies after transformation
and transformed ccc plasmid alone as usually incubating 1 hr in ice preor to heat shock and heat shock at 42 deagrees for 60 seconds and back to ice then suspending in LB broth and incubation at 37 for 1or one hour 30 min and plating in LB agar plates containing 50 microgam per ml concentarion kannamycin then over night incubation at 37 degrees
i used to get good colonied in contol for transformation i am getting good colonies
the rest of the plated not even single colony on agar plates containing 50 microgam per ml concentarion kannamycin
please give suggestion
thank you