plasmid DNA Digestion - (Jun/11/2007 )
Hi,
I ususally use 75ng-100ng digested and purified plasmid per ligation reaction and a vector:insert ratio of 1:3.
How much plasmid DNA do you use in your ligation reaction?
And how much of the ligation reaction you use in transformation?
I was thinking that if I need less DNA per reaction then I won't need to digest 1ug plasmid DNA like I usually do.
And if I have less DNA to digest, digestion will more efficient and quicker.
Thanks
I ususally use 75ng-100ng digested and purified plasmid per ligation reaction and a vector:insert ratio of 1:3.
How much plasmid DNA do you use in your ligation reaction?
And how much of the ligation reaction you use in transformation?
I was thinking that if I need less DNA per reaction then I won't need to digest 1ug plasmid DNA like I usually do.
And if I have less DNA to digest, digestion will more efficient and quicker.
Thanks
hello,
For the ration I use the same (1:3 vector:insert). The quantity of insert (Y) that I use (for the ligation) depend of the size of the vector and the size of the insert: Y=3* (( x pb of PCR product or digested DNA) * (50ng of vector))/ (size of vector in pb). So, I use alwas the same quantity of vector 50 ng.
For the transformation I use 2 or 3 µl of the ligation reaction (which is performed on 10µl)
I hope that this help you
I ususally use 75ng-100ng digested and purified plasmid per ligation reaction and a vector:insert ratio of 1:3.
How much plasmid DNA do you use in your ligation reaction?
And how much of the ligation reaction you use in transformation?
I was thinking that if I need less DNA per reaction then I won't need to digest 1ug plasmid DNA like I usually do.
And if I have less DNA to digest, digestion will more efficient and quicker.
Thanks
hello,
For the ration I use the same (1:3 vector:insert). The quantity of insert (Y) that I use (for the ligation) depend of the size of the vector and the size of the insert: Y=3* (( x pb of PCR product or digested DNA) * (50ng of vector))/ (size of vector in pb). So, I use alwas the same quantity of vector 50 ng.
For the transformation I use 2 or 3 µl of the ligation reaction (which is performed on 10µl)
I hope that this help you
Yes, it helped.
Actually, I knew how to calculate the amount of insert.
I just wanted to know how much vector you use. I think I have been using way to much. 100 ng vector in 10ul ligation reaction and I use everything (10 ul ligation reaction) to transform bacteria.
Maybe you will think I am crazy but the truth is that I have been tought like this and in fact always works but I just thought it would be more reasonable to reduce these amounts.
Thanks
I ususally use 75ng-100ng digested and purified plasmid per ligation reaction and a vector:insert ratio of 1:3.
How much plasmid DNA do you use in your ligation reaction?
And how much of the ligation reaction you use in transformation?
I was thinking that if I need less DNA per reaction then I won't need to digest 1ug plasmid DNA like I usually do.
And if I have less DNA to digest, digestion will more efficient and quicker.
Thanks
I usually use 50 ng of vector but will cut back to 20 - 25 ng, reducing the amount of insert and the size of the ligation rx. I then use 50 - 100 ul of competent cells and add no more than 5 ul of the ligation per 100 ul of cells (more can actually reduce the transformation efficiency). Finally I plate roughly 1/10 of the cells - keeping the rest at 4 dC until I see if I have enough colonies.
I find that most kits and protocols have you ligate way more than you need.....
Lauri
I routinely use 5 ng of vector and an appropriate amount of insert in a 5 uL ligation reaction. For transformation, I add 1 uL of ligation reaction to 25 uL of chemical competent cells. I add 680 uL of SOC, express for 1 hour, and plate out 30 uL to generate 300 - 500 colonies per plate.
Well,
I am satisfied with my results. I used ~50 ng digested and purified vector, ratio 1 : 3 vector : insert and trassformed bacteria with 2ul of ligation reaction ( 10ul final volume). I had lots of colonies in the positive control and almost no colonies in negative control.
Additionally, as I expected transforming bacteria with 2 ul ligation mix gave me much better results than 8ul.
Thanks