I have been trying to clone mCherry from another vector into pLVX. mCherry is flanked by a NotI site at the 5' end and a BamHI site at the 3' end. The two sites are present into pLVX contigous one to the other: 5' NotI-BamHI 3'. The restriction digest of mCherry woks fine, since I get a 751 bp product, as expected. In parallel, I digested pLVX with the two enzymes at the time, at a ratio of 4:1 units NotI:BamHI, using fermentas BamHI buffer, as recommended in the website . The digestion seems good, as compared to the undigested vector (undigested: 2 bands; digested, linearized vector). For the ligation , I am using the T4 ligase and the 10X ligation buffer from NEB, at various insert:vector ratios (5:1;10:1; 20:1;1:1) using 25 ng vector always. I have tried 1h ligation at 18°C as well as overnight. I am using TOP10 cells for the transformation (electroporation) or Stbl3 (heat-shock 42°C, 1'30''). I had previously cloned different inserts into this vector succesfully, but now I get no colonies at all in all of the combinations I have tried. What else can I try?
Hi Sebastian,
You should digest your vector with NotI first for a few hours and then add the BamHI and digest for a few hours. NotI requires a lot of bases on either side of its restriction site and so if BamHI cuts first then the required number of bases are not there for NotI to cut.
Good Luck!
Here is what you could do and what has worked for me, since the two sites are very close:
Digest 1 ON -> purify -> Digest 2 -> purify (optional if buffer compatible with CIAP or any other) -> CIAP -> Inactivate or purify (I know u might lose a lot of DNA, but it works)
Ligation 1:3 and 1:5 being 1=50ng vector I believe that more than 1ug migh block your competent cells. If your vector is a low copy incubate them more time and also try incubating at 30°C
Good luck brother
Hi Sebastian,
I'm not sure about how strict on incubation temp of the ligation. I have experienced on the ligation but I worked at 16oC for overnight as recommended in the protocol (while you set at 18oC), the result was good. So, have you tried to ligate them at 16oC? And how about the selectable antibiotic, did you use the correct one in appropriate concentration? I hope it will help, good luck.
Ligation protocol: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202
Thanks to all for your replies. Actually, we realized that one of the constructs it's quite dirty (there is a smear in the gel), so this might be one of the reasons why I can't obtain a succesful ligation. Apart from that, I will use 50 ng vector instead of 25ng, and I will try doing sequential digestions, precipitating after the first one, and then purifying from the gel.
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