Ligation problem again - (Jan/13/2009 )
Hi all
insert 2kb
vector 6.5 kb
both digested with smaI (blunt end)and gel extracted (vector not yet dephosphorylated, so can i dephosphorylate just before ligation??)
i ran out a little bit of my gel puri and it seems i just have around 4ng/uL of DNA (for both insert and vector)
so if i want atleast 20 ng of vector and at least 1:3 ration for vector/insert, i should use atleast 5uL of vector and 15uL of insert, doesnot this sound lot for a ligation raction
further if i want 1:8 ratio, i will have to use loads of insert
can i proceed with this DNA prep or do another digest with more vector DNA??
thanks
insert 2kb
vector 6.5 kb
both digested with smaI (blunt end)and gel extracted (vector not yet dephosphorylated, so can i dephosphorylate just before ligation??)
i ran out a little bit of my gel puri and it seems i just have around 4ng/uL of DNA (for both insert and vector)
so if i want atleast 20 ng of vector and at least 1:3 ration for vector/insert, i should use atleast 5uL of vector and 15uL of insert, doesnot this sound lot for a ligation raction
further if i want 1:8 ratio, i will have to use loads of insert
can i proceed with this DNA prep or do another digest with more vector DNA??
thanks
I don't think your calculations are right!! You need to rethink the whole thing!!
insert 2kb
vector 6.5 kb
both digested with smaI (blunt end)and gel extracted (vector not yet dephosphorylated, so can i dephosphorylate just before ligation??)
i ran out a little bit of my gel puri and it seems i just have around 4ng/uL of DNA (for both insert and vector)
so if i want atleast 20 ng of vector and at least 1:3 ration for vector/insert, i should use atleast 5uL of vector and 15uL of insert, doesnot this sound lot for a ligation raction
further if i want 1:8 ratio, i will have to use loads of insert
can i proceed with this DNA prep or do another digest with more vector DNA??
thanks
I don't think your calculations are right!! You need to rethink the whole thing!!
Once again,
based on my gel staining compared to ladder, i figured out that my insert and vector are both about 4ng/uL. so if i want to start with 20ng of vector i should use 5uL of vector and to get a insert/vector ratio of 3:1 i should use 15uL of insert. why is this calculation wrong??
insert 2kb
vector 6.5 kb
both digested with smaI (blunt end)and gel extracted (vector not yet dephosphorylated, so can i dephosphorylate just before ligation??)
i ran out a little bit of my gel puri and it seems i just have around 4ng/uL of DNA (for both insert and vector)
so if i want atleast 20 ng of vector and at least 1:3 ration for vector/insert, i should use atleast 5uL of vector and 15uL of insert, doesnot this sound lot for a ligation raction
further if i want 1:8 ratio, i will have to use loads of insert
can i proceed with this DNA prep or do another digest with more vector DNA??
thanks
I don't think your calculations are right!! You need to rethink the whole thing!!
Once again,
based on my gel staining compared to ladder, i figured out that my insert and vector are both about 4ng/uL. so if i want to start with 20ng of vector i should use 5uL of vector and to get a insert/vector ratio of 3:1 i should use 15uL of insert. why is this calculation wrong??
cause your insert MW is alrady 1/3 of your vector. So you use about equal ng of both. 5ul insert, 5ul vector. That makes it 3:1 insert/vector.
hi..
maybe you can try using CIP treatment for your vector...
so as to reduce self-ligation of a vector digested with enzyme to create compatible sticky ends
Hope it helps...