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should we dilute the DNA (vector and insert), before ligation? - ligation problem (Jun/22/2005 )

Hi.. i have trouble in determining whether i should dilute the insert and vector before i prepare the ligation mix. I'm not sure if it's accurate, but i've read a ligation protocol that said, if the concentration of the DNA is too high, the ligation process might fail. And is there anybody who's using NEB T4 DNA ligase? Should we dilute the T4 ligase and 10X buffer as well? For your information, i'm trying to ligate a 8 kb insert (digested with SpeI) to a 31.2 kb vector (which i've digested with AvrII). If there's any protocols that might work, can anybody send it to me...thank u soo much.

-alhana-

QUOTE (alhana @ Jun 22 2005, 08:40 PM)
For your information, i'm trying to ligate a 8 kb insert (digested with SpeI) to a 31.2 kb vector (which i've digested with AvrII). If there's any protocols that might work, can anybody send it to me...thank u soo much.


Hi,

do SpeI and AvrII produce same sticky ends? or blunded ends?

If not, how can you ligate incompartible ends?

I heard that too diluted ligation mixture is not good, but I am not sure about the aftect of too high concentration.

-dtle-

QUOTE (dtle @ Jun 22 2005, 08:57 PM)
QUOTE (alhana @ Jun 22 2005, 08:40 PM)
For your information, i'm trying to ligate a 8 kb insert (digested with SpeI) to a 31.2 kb vector (which i've digested with AvrII). If there's any protocols that might work, can anybody send it to me...thank u soo much.


Hi,

do SpeI and AvrII produce same sticky ends? or blunded ends?

If not, how can you ligate incompartible ends?

I heard that too diluted ligation mixture is not good, but I am not sure about the aftect of too high concentration.


I think you really dont need make dilutions if you do master mix, but you must guarantee the right concentration.

-donisaid-

QUOTE (dtle @ Jun 22 2005, 08:57 PM)
QUOTE (alhana @ Jun 22 2005, 08:40 PM)
For your information, i'm trying to ligate a 8 kb insert (digested with SpeI) to a 31.2 kb vector (which i've digested with AvrII). If there's any protocols that might work, can anybody send it to me...thank u soo much.


Hi,

do SpeI and AvrII produce same sticky ends? or blunded ends?

If not, how can you ligate incompartible ends?

I heard that too diluted ligation mixture is not good, but I am not sure about the aftect of too high concentration.



SpeI and AvrII has a compatible ends and i've checked the two enzyme's compatibility in NEB catalogue. The problem is, i'm not doing a master mix, that's why i want to know whether the dilution is necessary or not. Anyhow. now i have another problem. the plasmid that i've extracted (using Amersham's kit) going out of the well each time i try to load it. i really dont know what to do..can u please help me?

-alhana-

If you are extracting plasmid with kits, make sure the colume dry properly otherwise the residue ethanol in the wash buffer will affect gel loading.

When I have this problem I would add more running buffer which will help to hold down the samples. Hope this helps

-Muly-

Muly is right residual EtOH causes problems with gel loading the other possibility is there is residual genomic contamination this would also cause problems.

Have you considered dry loading samples??

With your initial problem I routinley don't dilute my ligations although I have had no experience using vectors so large.

Scott

-Scott-