Ligation of big vs small - (Jan/22/2006 )
Hello, need some suggestions here.
Considering that ideally we will ahve to have a ration between the size of our vector and the fragment we want to insert on it. 1:1, 1:3.. how would you manage a ligation of a 10 kbp vector with a 0.1 kbp fragmennt. For a final volume of 10 ul where 5 is used by the buffer (promega 5x buffer) I thought we would have to use something like 4.95 ul of the pcr fragment (which is the one am using ) and 0.05 of a theoretically very concentrated cut vector.
Does this make sense to you.. if it is.... let see if i get some colonies (a bit difficult I would say)
thanks
tertu
I don't think you have to worry about this, use more vector (use 1ul cause that is what you can pipette) and/or increase the total volume of your ligation and only transform a portion of the ligation mix...
promega T-easy suggests 1:8-8:1 range of ratios, and if you have a good way to screen it shouldn't make much difference, especially with sticky end ligations... If you are very concerned about the vector coming back together then dephosphorylate it before adding to the reaction, shouldn't be a problem...
If you REALLY want to keep this ratio then make up an appropriate volume (say 50-500ul) of solution with the correct insert/vector ratio and then ethanol precipitate together (you can concentrate them both this way too) then resuspend the pellet in an appropriate volume of dH2O and add to the ligation buffer/ligase...
btw--your calculation is off it would be: 5ul 2X ligation buffer + 1ul ligase + 4ul total DNA....
for 5X buffer you have even more room for DNA: 2ul 5X ligation buffer +1ul ligase + 7ul total DNA...
hope this helps you, good luck...
Thanks a lot Beccaff... clever idea to concentrate ... might give it a try
cheers
luis
promega T-easy suggests 1:8-8:1 range of ratios, and if you have a good way to screen it shouldn't make much difference, especially with sticky end ligations... If you are very concerned about the vector coming back together then dephosphorylate it before adding to the reaction, shouldn't be a problem...
If you REALLY want to keep this ratio then make up an appropriate volume (say 50-500ul) of solution with the correct insert/vector ratio and then ethanol precipitate together (you can concentrate them both this way too) then resuspend the pellet in an appropriate volume of dH2O and add to the ligation buffer/ligase...
btw--your calculation is off it would be: 5ul 2X ligation buffer + 1ul ligase + 4ul total DNA....
for 5X buffer you have even more room for DNA: 2ul 5X ligation buffer +1ul ligase + 7ul total DNA...
hope this helps you, good luck...
Considering that ideally we will ahve to have a ration between the size of our vector and the fragment we want to insert on it. 1:1, 1:3.. how would you manage a ligation of a 10 kbp vector with a 0.1 kbp fragmennt. For a final volume of 10 ul where 5 is used by the buffer (promega 5x buffer) I thought we would have to use something like 4.95 ul of the pcr fragment (which is the one am using ) and 0.05 of a theoretically very concentrated cut vector.
Does this make sense to you.. if it is.... let see if i get some colonies (a bit difficult I would say)
thanks
tertu
I have recently been performing blunt ended ligations with inserts that were either 240 bp or 2.7kb. I used the NEB Quick Ligase Kit which sounds similar to Promegas. I found the smaller fragments easier to ligate into the vector and a 100bp should cause you no problem. I performed the following reaction which was roughly a 3:1 ratio:
Vector (45ng/ul) 1.1
Insert 25ng/ul) 1.0
H2O 7.9
Buffer 2x 10.0
Ligase 1.0
I got colonies on all plates using the various 240bp fragments. In addition some people suggest increasing the 3:1 ratio but for my longer insert (2.7kb) this actually had a negative effect on the ligation.
Hope this helps