I am trying to perform a rather difficult blunt-end ligation that requires several enzymatic steps and would appreciate any tips from those who have experience with blunt-end cloning.
My protocol thus far has been as follows:
1. Restriction digest of backbone vector (7,600 bp) with one blunt-end and one sticky-end (5' overhang) RE. Gel purify fragment and clean with spin column.
2. Blunt backbone DNA with Klenow fragment. Purify with spin column.
3. Dephosphoylate backbone DNA with Antarctic Phosphatase. Purify with spin column.
4. Cut insert fragment (7,900 bp) from donor vector using one blunt end and one sticky end (3' overhang) RE. Gel purify fragment and clean with spin column.
5. Blunt insert DNA with Klenow fragment. Purify with spin column.
6. Perform ligation using 100 ng of 7.6 kbp backbone and and 1:1 or 3:1 molar ratio of 7.9 kbp insert DNA in a 10 uL total volume reaction using NEB T4 DNA ligase.
(I tried incubating this at 16oC overnight and also at 25oC for 1 hour followed by 1:20 dilution of reaction with 1X T4 ligase buffer and incubation at 25oC for 8 more hours as recommended on tip 2 in https://bitesizebio.com/19149/7-ways-to-improve-blunt-end-ligations/)
7. Cleanup and concentrate ligation reaction mixture by spin column and use to transform electrocompetent E. coli cells.
No luck so far. Any other tips for this protocol? For instance, should I phosphorylate the insert fragment following the blunting reaction using T4 PNK? Am I unlikely to succeed with this reaction at all given the size of the fragments involved? Any advice would be appreciated.
Looks tough but definitely do-able. I think if you had two clean large blunt end fragments (one de-phosphorylated), it would be "easy." Problem is you probably have two large blunt end fragments that are a mix of all the reaction intermediates (e.g., some sticky end fragments (not Klenow filled), some blunted but not dephosphorylated fragments, some blunted + dephosphorylated fragments (the desired product)). So, each of those can interfere with the ligation and block the ligation product you want. I think you can make the final product cleaner by making each reaction as efficient as possible. For example-- only start with 1-2 ug DNA. You will lose some on the way but let every reaction run to completion. Then, even though you may only have a little bit of product at the end, you will know it's clean. What do you think?
Sounds heroic. I would use the PCR to make both fragments.
- type II restriction to avoid blunt end?
Leila, I ran the blunting and dephosphorylation reactions according to the protocol for each, which involved using only 1-2 ug of DNA. It is my understanding that letting the dephosphorylation reaction run too long can cause bases on the end to be chewed off. I'm not sure if the same is true for the Klenow fragment blunting though.
Misha, I didn't even consider using PCR to amplify these fragments. Do you think it is likely I will be able to amplify a ~7.5 kbp fragment from a ~11.5 kbp plasmid?
Hi, I think your protocol is ok. I will sugest two tricks that helped me. First, you might use 100ng as the máximum total DNA for ligation and I would try also with 50ng as total DNA, with the molar ratio you already tested. Second, before you mix everything for ligation you should put your DNA at 40-45°C for 10minutes and inmediatly put DNA on ice to avoid secondary structure, also mantain everything con ice and only perform the reaction at 16°C.
I hope this help and if it doesn't you could clone the fragment in 2 steps using a restriction site in the middle of the insert.
Hi Christopher,
I would have to agree with Misha and use PCR to at the very least engineer in restriction sites. There is also a technique called Overlap Extension PCR Cloning that can be used to make the kind of construct you are describing without any digestion or ligation steps. See the reference below.
Article Overlap extension PCR cloning: A simple and reliable way to ...
PCR may help - but remember that it is quite error-prone, so you risk creating mutations.
You can go through KS+ vector instead of any commercial one. Although its small size, you can clone long insert easily. Cloning step: cut plasmid by EcoRV to produce blunt end, end fill by T4 polymerase, use 1:4 ratio for Vector to insert. finally, use any company's ligase and transform into DH5alpha. Once you can clone in Ks+ than it will be easy to sub clone in any other your interest plasmid. There are so much loss if you include many steps in molecular biology.
Hi Christopher
I have the same problem. Have you managed to do this cloning? Did you phosphorilate your insert? Any tips?
Thanks a lot!
Hi Bruna,
I tried many, many approaches, but I never got this blunt end cloning to work. From my experience, you would definitely want to dephosphorylate the backbone and phosphorylate the insert if, for instance, you are amplifying it by PCR (my insert was from a restriction digest fragment, so this wasn't necessary for me). My best advice is that if it is at all possible to go back to the drawing board and find a way to clone your construct that doesn't involve blunt-end ligation, then do that. It will save you so much time and frustration.
Look into possibilities such as using Gibson assembly or Golden Gate assembly. How I eventually succeeded at cloning this construct was to use Golden Gate assembly to create a version of my large insert sequence where recognition sites for the positional cutting enzyme BsaI were eliminated from the internal sequence by making "silent" single-nucleotide substitutions to retain the same amino acid sequence of the translation product. Then I used two BsaI sites at the ends of my insert to create sequence overhangs that were compatible with staggered-cut restriction sites on the backbone I was originally trying to insert into.
I hope this can be of some help to you. Good luck.
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