DNA ligation and trasformation - (Feb/06/2013 )

hello, i have performed ligation for my sample. the concentration of my vector is 8 ng/ul while insert is 15 ng/ul. i used 1:3 ratio of vector insert where insert + vector= 100ng..this sample done in 4,16,23 and 25^oC. The ligation product then stored in -20^oC and 4 ul of ligation product then used for transformation on dh5a. however i got no cloning culture. i used invitrogen t4 ligase..is there any problems in my methods?

You can try to dilute your ligation reaction (after it's done) before doing the transformation. I usually dilute my 5 ul rxn to 25 ul using sterile dd H2O then transform 4 ul. It can lower the salt concentration and increase the yield. Btw, how big is your insert?

We do ligation at 16 degrees and it works fine. The ratio of insert : vector depends on what vector we are taking. We also use T4 ligase ..so method should be fine.

Usually the problem is in DNA or the competent cells, not the ligation. Have you measured the competence of your cells? How are your DNA parts being prepared?

some information about ligation-

http://www.addgene.org/plasmid_protocols/DNA_ligation/

Is it a high copy number vector ?
Your ligation sounds fine to me
Is it because your insert DNA too toxic for the cell because of the high copy number vector?
It's just a thought

tq..
littlepumpkin:

Lisa Hsu on Fri Feb 8 11:53:53 2013 said:

Is it a high copy number vector ?
Your ligation sounds fine to me
Is it because your insert DNA too toxic for the cell because of the high copy number vector?
It's just a thought