Ligation issue: Does vector size matter? - (Jun/19/2009 )
perneseblue on Jun 26 2009, 03:12 AM said:
yes, as the vector becomes larger, the ligation does become more difficult. As mentioned by the Homwbrew, it is the probability of the ends of the ligated molecule meeting. When the ligated molecule is short, the ends of the molecule are effective close together. The two ends of a short linear DNA molecule can not wander far. Contrast this to a long linear DNA molecule.
In general, some difficult is first encountered once the ligated DNA molecule exceeds 10kb.
If you do gel extraction of your vector, try to avoid or minimize exposure to UV light. It really does reduce the probability of obtaining your desired plasmid.
In general, some difficult is first encountered once the ligated DNA molecule exceeds 10kb.
If you do gel extraction of your vector, try to avoid or minimize exposure to UV light. It really does reduce the probability of obtaining your desired plasmid.
thanx hanming86 ans perneseblue for your suggestions. In dealing with the ligation of the vector which is more than 10Kb in size, I was succeed with both sticky and blunt end ligation. The important points are, I think, the molar ratio (1:8 to 1:10 vector:insert with blunt end ligation and 1:3 to 1:5 with sticky end ligation) and the temperature (lower temperature normally works better for me). I'm happy because at least now I have the 13kb plasmid with pBR322 ori, which has been said to be difficult to excess 10Kb. In the other work, I'm going to construct other 17kb plasmid with the same vector. What a challenge! but now I have some experience.
By the way, can I ask for the ethanol precipitation method? I know that but I didn't use. Actually I tried once to condense my DNA but I lose it, then I didn't perform it anymore. But since many ppl suggest that, I think it's good, just the procedure I used might not correct.
@swanny: yes, i agree with you. Recently I saw a member ask for an article about the 2 step ligation. I can't download this paper but when I looked into the abstract, the general ideal is using the high DNA concentration in the first step to ligate 1 end of each molecule to each other, then the concentration is reduced in the second step, which is more preferred to have a succeed self-ligation. I wonder if there is someone tried it before to ask for the detail procedure. I myself will design the experiment base on this idea to see how far i can go ^^
-Quasimondo-