ligation transformation kit - fermentas kit (Aug/03/2007 )
Hi dear,
recently I recived a transformation ligation kit, but i dont know how can I use it, it was for 2nd time that I couldnt get colones, please help me,
farhad
Which kit is it?
fast ligation transformation kit from fermentas,
Is this ligation kit newly purchased? If kit is new and not mistreated, the kit should be fine. The fault probably lies elsewhere, which is where the trouble lies.
Can you tell us about your ligation? What is your insert? Is it a PCR fragment or fragment from another vector? If PCR, describe your primer(eg the primer sequence). Which restriction enzyme did you use? How long did you cut the fragments for? How did you clean up your DNA.
Did you dephosphorylate your vector? What are your dephosphorylation conditions? How much vector was dephosphorylated? How much DNA did you use? What ratio of vector to insert. What are the sizes of your vector and insert (bp)?
What is your ligation formulation? How long did you ligated? What was your clean up method if any? What kind of transformation did you use - chemical or electrophoration? What conditions/protocol did you use. Did you recover your cells - what markers do your plasmid contain?
Did you have any vector only control in the previous attempts? Do you know you still have DNA in your tube just prior to the transfromation, did you run a gel to on the post ligation mix to see that the ligation step was sucessful.
Phew... long list. But as you can see, there are many points along the way where things can go wrong. We need a detailed story and controls.. to pin point what has gone wrong.
this is ligation transformation kit, I have done ligation and transformation using regular methods and I have gotten many colones, only something that I wory about is storage time of my fragments, how is it important I stocked it in -20, it around 2 months, by the way I only need some comments about this kit how should I use it?
think
Can you tell us about your ligation? What is your insert? Is it a PCR fragment or fragment from another vector? If PCR, describe your primer(eg the primer sequence). Which restriction enzyme did you use? How long did you cut the fragments for? How did you clean up your DNA.
Did you dephosphorylate your vector? What are your dephosphorylation conditions? How much vector was dephosphorylated? How much DNA did you use? What ratio of vector to insert. What are the sizes of your vector and insert (bp)?
What is your ligation formulation? How long did you ligated? What was your clean up method if any? What kind of transformation did you use - chemical or electrophoration? What conditions/protocol did you use. Did you recover your cells - what markers do your plasmid contain?
Did you have any vector only control in the previous attempts? Do you know you still have DNA in your tube just prior to the transfromation, did you run a gel to on the post ligation mix to see that the ligation step was sucessful.
Phew... long list. But as you can see, there are many points along the way where things can go wrong. We need a detailed story and controls.. to pin point what has gone wrong.