I need to clone my small size insert of 33bp only (in vitro anneled duplex oligo) into Psicheck2 plasmid (6.7 KB) by single digestion with NheI, but my ligation is getting failed every time. I was using the 1:1 ratio of Vector: insert i.e insert is 0.5ng and vector is 100ng with overnight ligation at 16 degree. After transformation followed by single colony incubation and plasmid isolation by miniprep, I have digested the extracted plasmid with NheI but found only single band of vector (0.8% Gel) , insert's band was not found (in 3% Gel).
please suggest your opinion to troubleshoot this problem.
Thank you.
What you are getting is self-religation. You have to dephosphorylate the vector and phosphorylate the insert for this to work. Either use T4 kinase on your annealed oligos (I am assuming here you did not digest the oligos rather you ordered them in a way the create an NheI compatible overhang). Easier is to order oligos that are phosphorylated. Check this by sequencing and not digestion since finding a 33bp band on a gel is a big hassel.
P.S if you are column purifying the oligos at any point this will not work properly since the size is too short to bind with enough strength to the column hence will just wash out. Instead anneal them in T4 ligase buffer and use that directly.
You haven't really given us enough information. Are your annealed oligo's designed to leave the appropriate overhangs for ligation, or do you digest with NheI first? Is your vector dephosphorylated or just digested?
Lastly are you sure you will see a 33bp insert? That is a really small insert that is going to have very low signal, you might just not be able to see it. Can you digest with some other enzymes to release a larger band that would be shifted in size by 33bp? Or design a pair of PCR primers to look for the insert.
@Michael J. Benedik
Thanks for your response.
Yes my annealed oligo have overhangs of Coresponding Restrictions enzymes NheI so we didn't disgest the insert and my vector is dephosphorylaed also. After invitro annealing I have run the anneled oligos (approx 200-300 ng) in 3% agarose gel with Etbr at 50V for 10-15 minutes only and yes the band was clearly visible.
I think visualizing size shifting by 33 bp in gel will be very difficult.
If you just annealed your oligos and tried to ligate them directly then you will not get anything. Normal oligos do not have a terminal phosphate (as Hanna Alalam described) so if you also dephosphorylate your vector then there will be no ligation reaction at all.
Regarding my comment about size, yes you can see a 33 bp band if you load 200ng. But when you are digesting plasmid DNA it will be very hard to get 200ng of the insert size. Your plasmid is 6000 bp and the insert is 33, so it is about 0.5% of the total mass. To have 200ng of insert you would need to digest 40 ug of plasmid. When you run a mini prep you are probably digesting less than 1ug. So in addition to the ligation problem, I really don't think you will see your insert.
1. You probably need 1:10 or 1:20 ratio of Vector: insert for small insertion ligation.
2. You need a backbone only, no insert ligation as your negative control and only if this negative control shows very few or much less colonies than the insert ligation then you could be assured that the ligation works.
3. You could go straight with Sanger sequencing instead of doing digestion verification.
Di Xia, Thank you so much for your suggestion, have one doubt please help me to understand this. As you mentioned to use no insert as my negative control and fewer colonies in this negative control will be assured for ligation works. but my vector in this experiment is single digested (NheI) then CIP treated, isn't it that self-ligation/no insert ligation will show more colonies?
Neha .. CIP treatment dephosphorylate the vector, thus preventing its self-ligation. It is an essential step for your single digested vector. Only if both digestion and CIP treatment is complete, it will show few or no clone in the negative control. On the insert side, you need to use T4 PNK (T4 Polynucleotide Kinase) to phosphorylate the insert, so that it could be ligated with the CIP treated vector.
As others have pointed out, it is tricky to see the release of 33bp insert from the construct. It might be too late but I would recommend designing the new construct with an insert sequence showing a different digestion pattern so that you could easily distinguish the new construct VS backbone. Eg. make the NheI uncuttable after the ligation or add a new restriction site in the insert.
PS: I would highly recommend gel purifying the single digested vector to reduce the chance of none digested plasmid carrying over to your ligation reaction.
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