problems in ligation of pET28b(+) - Ligation (Oct/06/2006 )

Hello everyone, it is the first time I use the forum.

I would like to insert 1.5kb fragment into pet28b(+) using Nco1 and Xho1 site. The 1.5kb insert was first cloned into a TA and then cut with the REs. The pET28b(+) was also cut with the same REs. Then I do a ligation reaction using new england biolab T4 ligase. In the reaction mixture, i added 1uL reaction buffer, 0.9ul ligase, 50ng vector and desired amount of insert in 10uL reaction acrroding to the manufacturer's instruction. I allowed them to ligate for 4 hrs and overnight. The control i did was one with 50ng vector+ligase+raction buffer. Then i took 2ul reaction mixture into 50uL DH5 alpha for electroporation. However, no colony was found even in the control plate.

I think there should be no problem with the competent cell because many colonies can be found when i transform pure plasmid.

I don't know what happen~
Please give some suggestions!!

-Michelle Lau-

QUOTE (Michelle Lau @ Oct 6 2006, 05:36 AM)

I would to insert 1.5kb fragment into pet28b(+) using Nco1 and Xho1 site. The 1.5kb insert was first cloned into a TA and then cut with the REs. The pET28b(+) was also cut with the same REs. Then I do a ligation reaction using new england biolab T4 ligase. In the reaction mixture, i added 1uL reaction buffer, 0.9ul ligase, 50ng vector and desired amount of insert in 10uL reaction acrroding to the manufacturer's instruction. I allowed them to ligate for 4 hrs and overnight. The control i did was one with 50ng vector+ligase+raction buffer. Then i took 2ul reaction mixture into 50uL DH5 alpha for electroporation. However, no colony was found even in the control plate.

I think there should be no problem with the competent cell because many colonies can be found when i transform pure plasmid.

a few questions;

How old is the ligase??

how many times has your reaction buffer been thawed and frozen?

How was your positive control cut and purified?

-Jimmy_september-

The enzyme is newly purchased for two months.
The ligation buffer is also newly thawed as we will aliquote into small vol and dispose after use.

As the vector was originally with an insert, so i just cut them with the two REs and gel purified it. Hopefully, the desired insert and backbone were seen.

Actually, I tried another REs before, and using the original plasmid for RE digestion. As i doubt if the RE digestion efficiency was low, so i tried to cut a vector with insert this time, however, it didn't work again.~

Do you have any suggestion?Thanks a lot!!!

-Michelle Lau-

well I assume that the T7 promoter is kept uninduced. So, i guess the first thing would be to confirm that there is DNA in the tube just before performing the transformation. Run a portion of the ligation mix DNA on a gel with a small narrow comb.

I would also drop the volume of ligase used (0.5ul is more then enough). I have the worried feeling that the levels of glycerol in the ligation mix is close to inhibitory (5%). On a personal peference note. I would also double both the volume of the ligation in use and the amount of DNA in the mix.

-perneseblue-

Thanks perneseblue for your valuable suggestion. I will try and see if it works.

But I would like to ask do I still use 5uL ligase in 20uL ligation reaction.

Thanks a million!!

-Michelle Lau-

0.5ul T4 ligase to 20ul reaction sample is fine.

5ul ligase is far too much.

-perneseblue-