6 replies to this topic

[ahmadfathi]

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?

[littlepumpkin]

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?

[neuron]

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.

[phage434]

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?

[neuron]

some information about ligation-

http://www.addgene.o...s/DNA_ligation/

[Lisa Hsu]

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

[ahmadfathi]

tq..
littlepumpkin:

Lisa Hsu, on 08 February 2013 - 03:53 AM, 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

what you mean by toxic?
i really need a clearance, i'm still a newbie in this area
my insert size is 1.5k bp..