problem in ligation and transformation - (Apr/25/2006 )

you don't put the entire 20ul into one tube of comp cells, do you? more than 10ul can have dramatic effect on transformation efficiency

and, hey, for the QiaQuick kit...I have learned (from Fred; he's awesome) and tested that you get vastly increased yield if you heat your elution buffer (50-60C) and let it sit on the column for 2-5 minutes, then spin.

so, if it doesn't work, maybe try that next time?

*crossing my fingers for your clone*

nope. only add 5ul to my cells - am also trying 2ul this time to see if even 5ul is too much. Do you heat inactivate your ligase? Mine is from NEB and the insert doesn't say anything about heat inactivation being essential so I don't - could that be a factor too?

Neat trick on the elution buffer! I just elute with water though and store my DNA at -20 because I thought the elution buffer had salts etc. that could interfere with subsequent reactions? If not, then I will use elution buffer next time because I lose so much DNA! How long do you heat the elution buffer for?

um, I just heat up the buffer in an eppy, in a temp block a few minutes...just enough till it gets warm...then spot it onto the matrix and let it sit 2-5' at RT before spinning

I was using the elution buffer...then I switched over to water and I use the fragments right away, or within a day or so, just to be sure they're not hydrolyzing away. I do not use the buffer because I know someone who had bad experiences

I do NOT heat-inactivate my ligase; I use chemi comp cells. I would only worry about that if it were electroporation, which is more sensitive to such things

So you no longer use the elution buffer?

I figure the DNA will be stable in water if I keep it frozen. Didn't think dsDNA would hydrolyze so quickly in water?

I have no idea of the exact kinetics, should take awhile anyhow, but I sure don't want to find out the hard way

if the cloning fails, I'm typically not one to dig up old bits and pieces for the next try, because you can't always be sure exactly where the mistake happened anyhow; I just start over. It takes, what, a day and a half from PCR of insert, to ligation and transformation? it's just not worth it

so, when I make a fragment or I have some purified vector or something, I tend to use it pretty rapidly...the longest would be two days over a weekend, but I'll usually come in to continue the protocol anyways on Saturday because I'm impatient for the results

everyone does it differently and I ain't no guru, that's just what works for me....

oh, btw, I do not use the elution buffer. I saw some evidence on two occasions that it was inhibiting the ligation, and then I heard from someone else in my department that he had problems as well...and like everything else, it's just not worth it. works fine with H2O, why take the chance?

I have no idea of the exact kinetics, should take awhile anyhow, but I sure don't want to find out the hard way

if the cloning fails, I'm typically not one to dig up old bits and pieces for the next try, because you can't always be sure exactly where the mistake happened anyhow; I just start over. It takes, what, a day and a half from PCR of insert, to ligation and transformation? it's just not worth it

so, when I make a fragment or I have some purified vector or something, I tend to use it pretty rapidly...the longest would be two days over a weekend, but I'll usually come in to continue the protocol anyways on Saturday because I'm impatient for the results

everyone does it differently and I ain't no guru, that's just what works for me....

oh, btw, I do not use the elution buffer. I saw some evidence on two occasions that it was inhibiting the ligation, and then I heard from someone else in my department that he had problems as well...and like everything else, it's just not worth it. works fine with H2O, why take the chance?

10mM Tris, pH 8.5 is what they say is in it

I know that it shouldn't matter without any EDTA, there's no apparent reason for it. Perhaps the pH is a bit high and interferes with the ligase buffer activity, if you are adding whopping amounts of your purified fragment?

Hi,

I'm having troubles with the ligation of my 580bp insert into my 2600bp vector.
I have done the following:
200 ng Vector
100 ng Insert
5 minutes 65degrees
cooling on ice
1 ul T4 DNA ligase buffer
0,5 ul T4 DNA ligase

Then I put it by 16 degrees overnight and after that I transformed it in GM2163 Electrocompetent Cells (2ul ligation in 40ul cells) But there were no colonies!

I also want to ligate an insert of 1200bp into an 2600bp vector, Ive tried it by doing it the same as I told before but also here there were no colonies!

Is there anybody who can help me??
Thank you very much

Marloes.

P.S. Sorry for my English, it isn't my best language.

Generally, you want more insert than vector. Also, what's the purpose of the 65°C step prior to adding the ligase?