Ligation - (Apr/06/2010 )

Hello,

I am trying to ligate Il-8 (insert) and pIRES Ds Red(vector)

I got the IL-8 from Addegene (pUC-IL-8) so I desinged the primers for it.I included two restriction sites in them.These sites are:XhoI and SacII.

I cut the vector with the same enzimes (there's specific site for them in the MCS)

I PCR the pUC-IL8 and I got a band with the right size,almost 2kb.I cut it out ,gel extraction and digestion with Xho I and SacII.
I digested as well the vector,with XhoI and SacII.

I prepared two ligation with TAKARA T4 ligase ,and 2 different ratio, 3:1 and 5:1.

For each ligation ,I prepared 3 tubes con 3 different incubation times

1.- 24ºC for 3 hours
2.-37ºC for 1 Hour
3.-16ºC overnight

For transormation in TOP10 competent cells,from invotrogene company ,I used the 20 ul from the ligation.

I didn't get any colonie after the whole process.

I would love to hear your suggestions!!!

Thanks!

1) make sure your primers have 5' overhangs from the cut site. Your enzymes will not cut a fragment at the end of a piece of DNA.

2) Minimize UV exposure during gel cutting. Use long wave (365 nm) or blue light.

3) Do controls! Can you transform the target vector without an insert? You should dilute uncut vector to 10 pg/ul and transform 1 ul. Many colonies should result.

4) It sounded as if you were transforming with all 20 ul of the ligation. This is too much. Use 1-2 ul in 50 ul of competent cells.

5) Ligation at 37 will be unlikely to do anything with 4 bp overhangs. It is unlikely that the ligation is the problem, but much more likely that poor quality DNA or bad transformation is the issue.

6) SacII leaves only a 2 bp overhang, and requires two sites for good activity. It would not be my first choice for an enzyme.

have you checked if your insert contains additional SacII and XhoI sites?

Have you checked insert&vector on a gel prior to ligation? ...you can always expect Murphys Law if you skip that step

Its always a sign that something went really wrong if you do not get any colonies ...normaly there are at least some background colonies.

Do a positive control with a standard plasmid of known concentration to test your competent cells.

Regards,
p

I recently managed to get my cloning done successfully and apparently my problem was that my PCR products didn't digest well. So a friend of mine recommended using ClonJET kit from Fermentas, so what I basically do now is to insert my PCR product into pJET, then cut from there and re-ligate in to the main vector. This way when I cut from the first vector I am sure that my PCR product is digested completely.

Also reduce exposure time to UV to a couple of seconds.