Protocol Online logo
Top : Forum Archives: : Molecular Cloning

Ligation- what to do if DNA is not concentrated? - (Aug/16/2005 )

Hi,

I'm going to do a ligatioin. The insert is 800 bp and the vector is like 14Kp. I'll need to use 200 ng of vector and 35 ng of insert in the ligation step. However, after cutting with restriction enzyme and running on the gel and further purification, the DNA concentration is so low that it's not likely I can do a 10 ul ligation reaction. What will you folks do with this kind of situation?

-fcheng-

You could try a number of things, here are a few that I think of...

Mix together your vector and insert and EtOH precipitate, you may need a carrier such as glycogen then resuspend in a smaller volume and ligate.

I don't know how dilute your sample would be, if you are close then you could go ahead and do like a 20ul ligation, maybe even more, but I think you should add more ligase and/or something like BSA to improve the probability of both your ends coming togther with a ligase.

Don't complete the gel isolation step, use an in-gel ligation protocol (EZclone systems has a good one) You could even re-run a gel with the preparations you have, then you don't have to worry about the concentration, except that you have to fit it into the well...

hope that helps...

-beccaf22-

QUOTE (fcheng @ Aug 16 2005, 10:42 PM)
Hi,

I'm going to do a ligatioin. The insert is 800 bp and the vector is like 14Kp. I'll need to use 200 ng of vector and 35 ng of insert in the ligation step. However, after cutting with restriction enzyme and running on the gel and further purification, the DNA concentration is so low that it's not likely I can do a 10 ul ligation reaction. What will you folks do with this kind of situation?


I have had this problem before, every time I tried gel purification a significant amount of my digested DNA had disappeared. In the end I estimated that the concentration of my DNA was approx 3ng/ul. I based my ligation reaction on this. I used a 1:3 and 1:5 ratios of plasmid:insert, diluting the plasmid to 5ng/ul plasmid DNA in a total volume of 15ul. I kept the concentration of DNA Ligase the same as i standard ligations. This has worked for me every time and I have helped many people who have had this problem by using this. If you ave any questions do not hesitate to ask. I hope this helps.

-Liz-

hi,
i agree with LIZ..there is no standard vector to insert ratio..If the insert size is very small compared to the vector i always try to take 1:5 or even 1:10 ratio of vector:insert..the amount of ligase being same as in the usual reaction conditions..for such small sized inserts,typically for a 10ul reaction,i take just 0.5ul of vector and 1ul each of 10X buffer and ligase and the remaining 7.5ul of the insert..you can set up two ligation reactions one in 16 degrees overnight and the other at 23 degrees for minimum one hour..Usually cohesive end ligation works better at 16 degrees..but for me,both the temperatures work and so i go for 23 degrees as it is quicker..

-proteus-

Thank you guys.
I'll keep trying! laugh.gif

-fcheng-

If your DNA is in water, you can put it in a vac√ľum spinner for some time?

(don't do this with DNA dissolved in anything else than water, because you will not only concentrate your DNA but also any ions or whatsoever).

-vairus-

hi! laugh.gif i usually precipitate DNA with 2 volumes ice cold EtOH absolute. I incubate it in -80C for 30 minutes or -20C for 1 hour. After that i spin it at maximum speed for 15 minutes. I washed the pellet with EtOH 70% (the pellet is usually invisible if the DNA conc. is low). then i spin it again at maximum speed for 1 minutes. Next, i let the tube stand upside down until the pellet is dry. now, i add deion water or TE RNase to dilute the pellet (i usually add about 25-30uL). beware do not too much adding water because u want the concentrated DNA, don't u? well... good luck! wink.gif

-listya-

If you don't have access to a speedvac which is fast and awesome way to concentrate DNA by the way, you can try using a dessicator.

The speedvac from our facility broke recently so I jury-rigged a makeshift one. I put an open tube with my DNA in a vaccuum container with dessicant at the bottom and left it on overnight. I concentrated my 90 ul DNA to a salt and resuspended in 10ul for the ligation.

Mountainman

-Mountainman-