I need to ligate a fragment of 800bp to the vector. I have digested the vector with Xba1. I have added Xba1 sites to both F and R primers, and got the fragment by PCR.
Anyone can suggest me the best way please. I tried it many times, but failed.
1. If you have to use one restriction enzyme than dephosphorylate the digested plasmid as this will remove the 5' phosphate groups and prevent re-ligation of the vector to itself.
2. Ensure that you add a few extra bases (I usually add 7) at the 5' end of your PCR primers before the restriction site as restriction enzymes do not usdually bind efficiently to the termini of DNA molecules - adding the extra bases gives a more efficient digest.
Are you saying that you don't get colonies or don't get colony with correct insert?
1) From what i can see, if you are using just one restriction enzyme, this may encourage self ligation which will give false positive colonies, you can try to digest your plasmid using two different restriction enzymes to prevent self ligation.
2) For efficient ligation, the raw material has to be in high quality or without contamination, meaning that both inserts and digested plasmid have to be purified before use
3) Note on the molar ratio of the insert and vector that you use. If you are just using 1 insert, you can use molar ration of 1:3 up to 1:10 to increase ligation efficiency.
4) Other than that, the selection plate has to be prepared fresh to ensure that the antibiotic is still working.
Hope this helps!
1. If you have to use one restriction enzyme than dephosphorylate the digested plasmid as this will remove the 5' phosphate groups and prevent re-ligation of the vector to itself.
2. Ensure that you add a few extra bases (I usually add 7) at the 5' end of your PCR primers before the restriction site as restriction enzymes do not usdually bind efficiently to the termini of DNA molecules - adding the extra bases gives a more efficient digest.
Dear Noor Bahadar,
The two above answers are correct and you should considered them.
Could you explain more about your experiment condition like: ligation temperature, ligation time, competent cell preparation, how many colonies did you gain and etc.
I used two different RE for cloning of desired fragment. I purified my desired fragment from agarose gel and used 1:3 molar ratio (according to below paper) and incubated the ligation reaction for 8-10h at 4-8 oC. I used chemically competent cell (E. coli DH5 alpha) which was freshly prepared.
Cranenburgh, R. M. 2004. An equation for calculating the volumetric ratios required in a ligation reaction. Appl Microbiol Biotechnol, 65: 200–202.
best regards,
Dear Noor,
The above suggestions are indeed very good. There is one thing that you need to be careful as well. XbaI can be blocked by overlapping dam methylation. If you add GA in front of XbaI site or TC after XbaI site, the XbaI site will be methylated in mose E. coli strains.
Above suggestions are very good. If still does not work, you may consider to use high concentration T4 ligase and incubate overnight at 4C. Don't finish your ligation mixture for transformation. Keep some and add T4 ligase again and incubate at same condition.
thanks all for your kindness. I would like to go for dephospho rylation. Anyone can share a well defined protocol please.
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