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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

-kalim-

QUOTE (kalim @ Jan 13 2009, 11:49 AM)
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

I don't think your calculations are right!! You need to rethink the whole thing!!

-TanyHark-

QUOTE (TanyHark @ Jan 13 2009, 04:40 PM)
QUOTE (kalim @ Jan 13 2009, 11:49 AM)
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

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??

-kalim-

QUOTE (kalim @ Jan 13 2009, 04:56 PM)
QUOTE (TanyHark @ Jan 13 2009, 04:40 PM)
QUOTE (kalim @ Jan 13 2009, 11:49 AM)
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

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.

-TanyHark-

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...

-galcrazy-