I have a problem ligating a small insert with larger vector. The ligation sites are XhoI and AgeI. After the double digestion I excised band for I and V and extracted DNA by glass milk, purified with Phenol/Chloroform and so on. Ligation was done by using 2x Ligation Kit, or by Ligation buffer/Ligase. I have optimized amount of DNA, stoichiometry of I/V (10/1). However, after transformation into DH5Alpha I got no colonies . Then I have another idea of utilization of DH10 beta derivative of NEB, I got colonies but the digestion assay of targeted plasmid suggested that I have a wrong product. SAD and I know that it is somehow and somewhat difficult to ligate, especially small Inserts with large plasmid. Appreciate with your tips!
Before doing ligation, did you treat the digested vector with the antartic phosphatase? it helps to avoid the self-ligation of the vector.
Thanks for your TIPS, but nonetheless what made me worry more is self-ligation of inserts (AB,BA,AB, probably). Importantly, I used two different restriction enzymes to cut V and I (AgeI/XhoI). This time I have tried with different molar ratio (I/V) for ligation overnight at 16 oC by using Ligation Kit. I have not killed enzymes after ligation reaction by heating up 65 oC as previous time coz Ligation Kit contains phenol which may impair transformation efficiency by after heating. I used 5 uL raw re-ligated plasmid DNA to transform into NEB10 beta competent E.coli (quite recommended for large size plasmid). Perfectly I got several 10 colonies and after prep., checking ligation, orientation...etc I got my right re-ligated product..... Happy and now transformation into host cell and await if cell can express protein, and if protein works as my expectation....
Hi Kien Xuan,
still interested?
Check In-fusin cloning from Clonetech. In this case you would not even need to do PCR - just order two or, more realistically, three long overlapping pairs of complementary primers, and add to the reaction. Works well.
Best,
Dmitri
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