I assume that you cloned your gene via restriction sites. If so, then you can cleave your ligated vector with the respective enzymes and release your insert. This insert should have the correct size. Additionally, you can cleave with two restriction enzymes, one of which cuts only in the vector and one of which cuts only in the insert. This way you can check whether you actually have an insert and you get an idea whether it is the right thing. In any case, if your digests look promising you will absolutely have to sequence your insert, even if you are dead sure that you cloned the correct fragment. This is because by the PCR that you employed to amplify the gene from E. coli might have introduced mutations (even polymerases such as Pfu produce errors every once in a while). You do not want to find out about that after you worked on the construct for a few years ... ;-)

I assume that you cloned your gene via restriction sites. If so, then you can cleave your ligated vector with the respective enzymes and release your insert. This insert should have the correct size. Additionally, you can cleave with two restriction enzymes, one of which cuts only in the vector and one of which cuts only in the insert. This way you can check whether you actually have an insert and you get an idea whether it is the right thing. In any case, if your digests look promising you will absolutely have to sequence your insert, even if you are dead sure that you cloned the correct fragment. This is because by the PCR that you employed to amplify the gene from E. coli might have introduced mutations (even polymerases such as Pfu produce errors every once in a while). You do not want to find out about that after you worked on the construct for a few years ... ;-)

Or, if your insert introduces a unique restriction site, you could cleave with that enzyme and a vector-specific enzyme.

Are you trying to check the ligation product before transformation? The best thing to do is transform it and then perform mini-prep on colonies from the plates with appropriate number of colonies.

For example, you ligate using 0ul, 1ul, 2ul, and 4ul of insert then transform into different E.coli preps... then check colonies and see if there is a bell curve from 0 to 4ul of insert. If so, take colonies from the 1ul and 2ul plates, do miniprep and restriction digest to release the potential insert. Successful ligations will have a band the size of your PCR product as well as a band for your vector. Unsuccessful ligations will likely have just the vector band.

I've attached a picture of a miniprep showing some successful and unsuccessful colonies. Lane 1 is the size marker, while lanes 6, 7 and 13 are unsuccessful ligations since they only have the vector. All of the other lanes have the insert being released and therefore are successful.

For a simple way...take a vector specific primer with 3' end close to your insert and take another primer used to amplify your insert in such a way that you could get a amplification with PCR and done. If there is product from this PCR then you got your insert. Size of amplified product will assure you for correct orientation (for blunt end ligation) as well as correct insert in vector.

OR

Use two vector specific flanking primers across cloning sites and you get PCR product with insert+vector back bone size.

Good luck

Using flanking primers specific to vector. You can use 3'primer from vector site and 5' from from cloned gene. You can also reverse it

Use vector specific primers like M13 Fow and reverse to check the insert in MCS region.

As mentioned above, by restriction digestion but of course you have to check the sequence and orientation. Even if you use High-Fidelity DNA Polymerase there is a chance of getting error.

I agree with Ralf's suggestion 100%. Instead of wasting time on other possibilities I would rather sequence my insert once I confirm the correct size by either double restriction digestion that would potentially release the insert from vector or by doing a PCR using the insert sequence-specific primers.

Hi! Mr. Dutta, you cut your gene with the internal sites. Check the enzymes present in the gene which serve as cutter for your gene by restriction mapper or NEB Mapper. Then set up an reaction using any one or two of that cutter enzyme of your gene. You calculate the band sizes and make conformation of your possitive clone.

Good luck.

you can use the appropriate restrictions enzymes to cleave Vector and DNA insert if you obtain 2 corresponding bands in the gel that proves that your insert is in the plasmid

Colony PCR. Design primers that will pull your insert out of your vector. Grow up 3-5 mL cultures of selected colonies. Take 5 uL from each and add to a PCR reaction. Give it a 5 minute denaturation on the first cycle to lyse the cells and after that just the standard set of cycles. Run the reactions on a gel, miniprep the cultures that came back positive. But, as others have suggested, do send your purified plasmid out for sequencing. Weird things do happen in cloning.

Dear Rebecca Montange

Do you mean that you do normal pcr with all steps including denaturation analing and extension. Then, you choose which colony has the correct size of your insert to choose to midiprep? is that right?

Yeah. You destroy the cells when you start the PCR so you need to start the 3-5 mL cultures first.but once you've found good colonies you can prep them. The nice thing is, because PCR is famously sensitive, you can take the samples hours before the cultures are ready for prepping.

you can confirm the presence of gene within the vector by two ways either by restriction by choosing the restrictions sites present in the primers if available of by using the restriction enzymes present in vector backbone or say mcs. or by pcr confirmation with gene specific primers or the either forward primer of vector backbone and reverse primer of in gene or vice versa. In all the case the if you get the expected size of band then gene is present else it is absent

good luck

You can either use m13 f and r primers or your own specific primers to amplify your gene of interest.

An earlier step like colony pcr could be done before plasmid purification to save time and materials.

I think a classical way by restriction enzyme mapping is the easiest. and the quickest, given that you have enough material. Choose the enzyme or combination of enzymes which will give you the fragments of different lengths for just vector and vector, containing insert, digest both, run on the gel next to each other along with MW controls and see whether there is a difference in the clones from the plain vector.

Another smart way to clone in a vector in is to design a restriction site which will be different after cloning compared to before cloning (creating or destroying a restriction site), so that after you do the cloning, one-step restriction would point to the vectors with the insert.

I think you can find the unique enzyme in your target sequence and then do some enzyme digestion experiments, in this we you can assure whether the sequence is ligated successfuly```

Colony PCR is the popular way. I usually design a pair of primers at the both end of the insert sequence, the primer also partially include some vector sequences. With this we can verify the size and the orientation of the inserted sequence.

However, we usually sequnce the constructed plasmid to make sure no mutation occures in the cloning process.

Colony PCR works very well, and is a quick check (about as quick as restriction analysis, and in some ways more convenient and sensitive). I usually design one primer specific for the backbone vector, and another internal to your cloned insert. I pick colonies directly into a PCR tube, and then spike into a 3-5ml tube of medium with appropriate selection. Once you've run your PCR, you can prep the positive culture(s). As Rebecca mentions above, add a 5min denaturation step at the beginning of your PCR program in order to lyse the cells.

Besides colony PCR and restriction digest, which has already been recommended in this thread, you could use a vector with a lethal gene that is disrupted by ligation of an insert into the cloning site, e.g. one of the pJET vectors from Thermo Scientific. In this case, only cells with recombinant plasmids are able to grow and form colonies.

Design your cloning strategy so as to remove one of the restriction sites of the vector that lies between the two sites chosen to insert your fragment and that is also absent from the fragment. After ligation, inactivate the ligase (10 min at 65 °C) and treat the ligation mixture with the restriction enzyme whose site should have been removed from yhour construct and transform. Positive transformants can be identified by colony PCR and further analyzed by plasmid miniprep and sequencing.

Easy way is to transform ligation product and if you have designed restriction site in your clone then after doing a miniprep(plasmid purification) for 4-5 colonies you can digest with the required enzymes and see if the insert release is of same size(as you have designed) and vector. Or you can take the vector and insert sequence and find the restriction site which is unique in insert and can digest with that if your plasmid linearlize then you can be sure for insert.

PCR with specific primers for your insert and vector can confirm your correct insertion. Howver, more reliable (my own experience) is a specific digestion with endonucleases and comparision of the theoretic digestion pattern with observed one.

I agree with Yaliang Ji ·, you may need to exam the product of your new gene.

Hi,

Colony PCR is a quick way to check your insert; you can check your insert by restriction analysis, most commonly done by using the same REs that you have used for cloning. However, sequencing is always recommended since PCR may introduce a mutation that may change the way your insert behaves in subsequent experiments.

I will go with Mr. Ralf Max Leonhardt as thats the way to check the insert which i follow too.

The safest is to sequence your gene, although the proof-reading capability of polymerases is very good a single base pair mutation could result in an inactive protein that you won't detect by restriction digest or PCR. You could waste weeks/months working on a mutated protein - a waste of time and money for the sake of a few hours for a DNA sequence.

As people have said, do a colony pcr or RE, preferably with two, one for the vector and other for insert.   

1) do a colony PCR with a forward primer for the vector and reverse for insert . 

2) do a digestion of your clone and empty vector and run them on a gel . ideally your clone should give a pop out of your insert 

3) if your insert is of significant size say above 500kbp, running both the empty vector and the clone on a gel against a ladder , you should be able to see the difference.  

i would still recommend sequencing just to make sure there is no mutation. If you are amplifying with Taq  then there are chaces of mutations because Taq has no proofreading capabilities. You should then go for a high fidelity enzyme . 

You could use a plasmid whereby you select for transformants  via a functional screen such as blue white colony selection. once you have identified your putative transformants, I would agree with Sharma, the colony PCR is fast and should give you the desired result. You should ultimately sequence for more definitive results.

Try performing some restriction digestion using enzymes which were used for cloning into plasmid or if you know the gene sequencing use few sets of  restriction enzymes to digest the gene and look for specific size bands on the agarose gel.

Colony PCR may tell the presence of insertion but not the orientation of ligation. Restriction digestions are the better method after sequencing-I think.

Personally I would prefer to do a colony PCR at first. Additionally you can check correct insertion by restriction endonucleases. Just look for nice restriction sites which gves you one or more nice visible fragments in gel. Nevertheless I would also sequence the gene if it should be used for downstream analysis. Because it can always happen that a polymerase error occured in amplification process - even with a prrofreading polymerase. Good luck!

For me the best way to quickly check positive transformants is to design an insert specific primer + a vector specific primer and make colony PCR. Understandably I never have positive control plasmid, but I always have some new positive clones which give PCR product of proper length :-) But  I alwaysseque nce my positive clones because of mistakes which can be made by polymerase during amplification of the  insert. Good luck!!!

The colony PCR or PCR from purified plasmid DNA using vector plasmid primers like M13 or T7 will give you the first idea about the length of the inserted product. As example, if only primers or smaller products were inserted you will reach a positive colony but a PCR product of about 150-200bp. Depending of the size of your product will be the size of the insert plus the small aplicon reached with the plasmid primers.

Best regards

Ckeck this page:

https://www.thermofisher.com/uk/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/5-ways-screen-recombinant-clones.html

Primary test: Colony PCR, Plasmid PCR and restriction digestion

Final confirmation though sequencing.

I think you are checking it after the ligation and not after transformation of the ligation mixture into the host.

You just can not check weather your insert has been inserted into the vector, as in ligation mixture, there are still a lot of other DNA material from your digested PCR and vector, which has not been ligated.

Its better to first transform the ligation mixture into a host and then do colony PCR or Plasmid isolation followed by RE digestion to verify your positive clones.