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Blunt-end ligation: what if the vector and insert are both phosphorylated?


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4 replies to this topic

#1 Curtis

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Posted 16 September 2018 - 10:35 PM

I have always used either vector with phosphorylated ends, or insert with phosphorylated ends when I do blunt-end ligation as at least one of them must be phosphorylated but I have never tried ligating vector and insert that are both pohosphorylated. Has anybody tried this?

#2 pito

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Posted 17 September 2018 - 07:11 AM

I have always used either vector with phosphorylated ends, or insert with phosphorylated ends when I do blunt-end ligation as at least one of them must be phosphorylated but I have never tried ligating vector and insert that are both pohosphorylated. Has anybody tried this?

 

I never really did this myself but if you check the data from commercial companies that mention information about blunt end cloning they always tend to write: "at least" the vector or the insert needs to be phosphorylated. (you said it yourself too: at least).

So I assume that if both are phosphorylated it does not hinder the cloning.

But as said: I never really gave it much taught since I just digest my plasmids with a RE giving me phosphor groups anyway.

 

I am kinda curious to know as well if others tried this. 


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#3 OldCloner

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Posted 17 September 2018 - 09:03 AM

Hi Curtis,

 

It is OK to have phosphorylated ends on both the vector and insert- BUT you will likely have a lot of “background,” meaning vector that has re-circularized without gaining an insert. Dephosphorylation prevents the self-ligation of the vector. High background is acceptable IF you are using a vector with blue-white screening capability on medium containing X-gal/IPTG (and a host strain that supports this). You look for the white colonies among the blue ones because the white ones have had the vector’s beta-galactosidase gene disrupted by your insert.

 

If you don’t have such a vector it is strongly recommended that you dephosphorylate the vector fragment before ligation, so there will be many fewer “background” plasmid-containing colonies.  Otherwise you will have to do a lot of screening to find one with an insert.  (Colony PCR can help make the screening more manageable if you must do it this way.  PCR can help screen for the proper orientation of your insert, too.)



#4 Curtis

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Posted 17 September 2018 - 10:47 PM

Thank you. I usually use pJet1.2 for my everyday blunt-end cloning and it does give self-ligating vector background so I usually pick many colonies to find the one with insert, as well as the one with the correct orientation.

But in a separate project that I am running I want to synthesize a polylinker and ligate it to a vector that has been double digested meaning the vector is already phosphorylated at the 5'-end. So before submitting the order to the company I wanted to know if I should order the linker with phosphorylated ends or just go ahead without it. According to your comments it looks like there is no need to have the linker phosphorylated.

Or alternatively I can order it phosphorylated and use SAP on the vector to avoid self-ligation. But honestly I prefer the first method as I am used to it and by experience I have seen that after picking several colonies at least one contains the insert. I am also not sure if SAP will efficiently remove all the phosphorylated ends. I have never tried it.

Thanks again.

#5 OldCloner

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Posted 18 September 2018 - 10:56 AM

Looks like pJet1.2/blunt is a specialized vector with a lethal gene (rather than a gene that makes blue color) to eliminate most of the background clones. That is an important piece of information. So what vector are you using for the new polylinker?

 

If the vector you are using for the polylinker does not have one of these lethal genes then you will get a lot more background than you are used to. Personally, I’d phosphorylate the linker and dephosphorylate the cut vector.  You are making yourself a lot of extra work in the screening for the tiny little insert.  I used to be a little worried about SAP, too, but switched to a recombinant Roche product called “rAPid” alkaline phosphatase and I have been very happy with it.  It works well on both sticky and blunt ends (follow instructions) and is easily heat inactivated. (No, I don’t work for Roche). 

 

When you do your transformations I assume you do the “no insert” ligation control along with your vector + insert reaction.  You should get very few colonies on the control plate and that will demonstrate that your background is low. You might not have bothered with this control for the pJET1.2, but you should if you do not have a lethal gene to get rid of (most of) the background.

 

ALSO- just thought of this- sometimes tiny little inserts do not interrupt the lethal gene (or beta-gal gene) enough- you can get some read-through especially if the reading frame is the same. So keep that in mind for your polylinker.






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