No colonies in my ligation - (Apr/09/2006 )
Hi everyone I have read a lot of these posts and have found the replies incredible helpful with other problems I have had in the past. I am having a lot of issues with getting ligations working in my lab. Here is my problem I am doing a double digestion overnight at 37 degrees C using HindIII and XbaI for an insert of 1.3kb and pcDNA3.1 (5.4kb). I generally use 2ug of DNA for both insert and vector. Afterward I run on a 1% gel and gel purify using Qiaquick columns. I then take 5ul of the insert and vector and run on a gel to make sure I have nice clean bands. Generally I then spec the sample and procede to ligation using a 3:1 insert to vector ratio, but since people have said UV spec are not reliable I have been eyeballing my DNA conc from the 5ul gel. I ligate using Invitrogen T4 ligase and transform with DH5a cells. I then get no colonies. I have checked competent cells, and the ligase and they both work. In the past I have used the qiaquick columns and they have worked. Somebody in a lab next to mine was able to get the ligation to work using her materials, but when I used her materials it didn't work. I am going completely batty and my boss is slowly losing patience since this has been going on for 4 months I really need to find the problem out. Please please lend me some advice.
Check that you are using long wavelength (365 nm) UV for examining your gels, and that you are exposing the gels to UV for the minimum amount of time.
you could try doing a PCR on your ligation mix (after you have incubated it) using a primer specific for your vector, and one for your insert - this will tell you if the ligation is working at all......and whether or not you need to check your cells.
I have found in the past that some cells just don't like some sequences - could you try XL1blue or JM109 instead?
It has worked! I tried using another UV box and I also increased the total amount of DNA present in the soln. I didn't get many colonies, but 7 out of the 8 I picked had the insert. Beggars can't be choosey I guess. Thanks for the help.