Question

When is it necessary to purify your DNA during cloning?

I cleaved a vector with two enzymes, after which I would like to blunt the ends and dephosphorylate before ligating to an insert. The insert was PCRed out from a different vector and then the ends were cleaved with an enzyme that creates blunt ends. I would like to reduce the purification steps to a minimal so that I can conserve the amount of DNA I have. Below is the process that I believe would be sufficient, please let me know if there are any additional purification steps that are required or if there are any I can skip:

Vector: Double digest -> gel purify -> Blunt the ends -> dephosphorylate -> ligate
(if I am using an alkaline phosphatase for the dephosphorylation I believe I can inactivate it and therefore skip the purification step before ligation?)

Insert: PCR -> gel purify -> restriction digest -> gel purify -> ligation

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  • Ilya Vainberg Slutzkin · Weizmann Institute of Science
    Hi,
    When considering purification steps you also need to take into account whether or not the following step can be performed in the same buffer. This might be a problem when doing the blunt ends -> dephosphorylate -> ligate steps for the vector.
    For the insert, if during the restriction you are cutting off only small fragments, you don't need to gel purify (since you already gel purified after the PCR), you can do a regular DNA concentration and purification with a kit.
    Best,
    Ilya.
  • Abdelhalim Boukaba · Chinese Academy of Sciences
    Hi!
    Is it absolutely necessary for you to make blunt ends cloning? Why not to perform cohesive ends cloning by adding restriction enzymes to your primers which are compatible with your destination vector?
  • Sarah Fazal · Kent State University
    Thanks for your responses.

    Ilya: So you're saying that if my buffers are different (which they are), I would need to include additional purification steps after the blunting and the dephosphorylating?

    Abdelhalim: Yes it is necessary to do blunt end cloning as there are no enzymes that cleave in the correct region of my vector that also do not cleave somewhere in the insert.
  • Ilya Vainberg Slutzkin · Weizmann Institute of Science
    Hi Sarah,
    I believe you should add those purification steps. It can be a simple DNA concentration and purification with a kit.
    You might be able to avoid some of these purification steps if the enzymes can be heat inactivated and the next enzyme can still work in the same buffer. For example, some NEB enzymes (like their alkaline phosphatase) work in some of their restriction enzyme buffers.
    Best,
    Ilya.
  • Ana Correia-Branco · University of Porto
    Hi sarah and Ilya,
    I usually do the dephosphorylating after any enzyme restriction (except Eam1105I), because CIAP (alkalyne phosphatase - which is the enzyme I use for this purpose) does not require a specific buffer. Usually works fine in any buffer. I further procced with the ligation reaction or, if necessary purify from gel.
    Relatively to the ligation reaction, note that blunt end cloning is ususally less efficient then with cohesive ends, so you should let it overnight (at least 12-16h) at 16ºC.
    Good luck.
    Ana
  • Aliona Bogdanova · Max Planck Institute of Molecular Cell Biology and Genetics
    Hi Sarah,

    i would recommend you:

    1) vector preparation: digest, blunting in the same buffer with addition of NTPs, change the buffer on buffer exchange column (Qiagen pink columns are my favourite), de-phosphorylation, and now gel purification, because it is very difficult to kill phosphotase;
    2) insertion: PCR, gel purification, digestion and buffer exchange with columns. You do not need to run another gel here, you just need to kill restriction enzymes and remove salt.
  • Luciano Martelotto · Memorial Sloan-Kettering Cancer Center
    Hi Sarah,
    Here is my suggestion. ALWAYS you digest a vector you should do a gel-purification step. Some people will say that they don't do it because it's a waste of time and that is ok, however, if you do it you are practicing good molecular biology as defined by Sambrook&Maniatis, and these guys knew something about it. Therefore, I will encourage you to do this step as often as you can, this will ensure you have no traces of undigested vector that will interfere during your transformation step (e.g. false positive) and consequently save you time later. The best way to save time is to do it right. You also want to get rid of buffers and ions that may (or may not) interfere with your phosphatase steps and have a clear start too. The later is not so critical though, as most reagents are now quite compatible with each other as well as between companies, which is great. Then, after phosphatase treatment the cleaning step depends on two things: if you used CIP (NEB), this enzyme you MUST gel-purify (recommended) or use a kit to get rid of the CIP that tightly binds to the end of your DNA and cannot be heat inactivated. If you used TSAP (Promega) 0r any other thermosensitive phosphatase, then you can heat inactivate it. The next thing to take into account is to check the buffer compatibilities between your recently 'phosphatased' vector, your digested construct and your ligation buffer. Most of the T4 DNA ligase in the market are compatible with any of the buffers for restrictions and phosphatase. For your Insert in the experimental setting you have provided, I believe you can skip it.
    I hope this helps!
  • Michael Bradbury · Alabama College of Osteopathic Medicine
    Sarah,

    One thing to considedr is how you can organize the steps to maximum benefit. I would do gel purigfication as the last step before ligation in both cases, and not do it until then. There is no reason to gel purify the insert twice, and I can see no purpose in doing it earlier in the process for the vector. Using a phosphatase that is easy to denature is a good idea. If you do try ligation in a restriction buffer, remember to add some ATP! That is missing from those buffers and included in ligase buffers for good reason.Blunting the ends can be done by a variety of methods, Klenow filling, T4 cut-back, etc.That is another place to consider whether you can just add enzyme and dNTPs to your current buffer. Remember, if you can do that, there is no need to do any processing except in a case where you would increase glycerol to more than 5% of your reaction, in which case heat-inactivation of the restriction enzymes would be advisable.
    Regards,

    Mike
  • Ria Goswami · Southern Illinois University School of Medicine
    Hi, the vector looks good.For the insert after doing PCR, you can run 3ul on a gel and see if it has the band of your interest and if there is no additional band other than that you can just treat it with DpnI to get reed of the template, column purify it, do restriction and then column purify again , that would be good.You do not need to gel purify everytime.
    Hope it works for you
  • Delphine Richer · French National Centre for Scientific Research
    Hi,
    I just add some details about the gel! It is very important to determine good percentage of your agarose gel. Because, sometimes, you can have products aspecifiques of very close PCR length of it of your product of interest.
  • Ilia Georgia · Medical University of South Carolina
    For me is difficult to do cloning with blunt ends.

    My advice is use Quick side mutagenesis kit. Order primers and make restriction sites in your plasmid any place where you like. By primers you can make restriction sites to your insert. After that you can digest plasmid and insert and do ligation and transformation.

    Quick site mutagenesis kit price is 150$, reaction takes 3-4 hours->transformation->miniprep and digestion. you need 2 days and cloning will go perfect.

    Best regards
    ILIA
  • Katarina Davalieva · Macedonian Academy of Sciences and Arts
    Hi Sarah,

    By my opinion it is absolutely necessary to include the purification step after the blunting reaction. I recommend the MinElute Reaction Cleanup Kit (Qiagen) which has great DNA recovery compared to classical ethanol precipitation method.
    If you are dephosphorylating with SAP there is no need of additional purification because SAP can simply be inactivated by heat step and do not interfere at all with the ligation process.

    Hope that helps,
    Good luck
    Katarina
  • Li Deyang · Fourth Military Medical University
    Hi. Sarah
    According to my experience, you do not have to do gel purification but just need to use PCR product purify kit to recover insert and vector, because cloumn can not recover DNA fragments less than 100bp.
  • Haitong Hou · Columbia University
    PCR with Pfu or Phusion will give you blunt end, and you only need gel purify PCR product.

    For vector, I will do
    Double digest -> purification from solution>Blunt the ends -> dephosphorylate -> gel purification> ligate
    gel purifcation before ligation is critical.
  • Ilya Vainberg Slutzkin · Weizmann Institute of Science
    @Li Deyang, the size of DNA fragments recovered by the column depends on the kit used. I used qiagen kits which recovered 60bp and even smaller fragments.
  • Li Deyang · Fourth Military Medical University
    @Ilya Vainberg Slutzkin: We only use OMEGA during vector construction. Qiagen...it is too luxurious. :p
  • Vidhu Sharma · University of British Columbia - Vancouver
    I would heat inactivate the enzyme and ethanol precipitate it rather than gel purification (if I do not see any non specific DNA material on the gel). The gel purification kits available add lot of junk to DNA besides very low recovery. Both these factors affect the subsequent step i.e ligation. Also, agarose is a very strong inhibitor even if in traces for ligation step. I tend to minimize gel purification as much as possible to reduce these additional contaminants introduced. Simple salt and ethanol precipitation has worked wonders for me in all my cloning projects. Good luck....
  • Antonio Parrado · Hospital Universitario Virgen de la Arrixaca
    I agree with Abdelhalim. Cohesive ends cloning with two enzymes is very easy compared with blunt ends cloning. A way of making it possible in case that the vector have not appropriate restriction enzyme sites in the MCS is introducing new sites by PCRing the whole vector.
  • Karthigeyan Dhanasekaran · Jawaharlal Nehru Centre for Advanced Scientific Research
    Always do a gel extraction after digestion and CIP treatment. You can only increase the starting amount of DNA to take care of the loss in subsequent steps. In case you are planning to do a drop dialysis after restriction digestion is also fine but after CIP treatment it is a must. I strictly prefer Gel extraction in both the steps. 50ng of insert is good enough for setting a ligation. All the best.
  • Simon Fellgett · Cardiff University
    Hi Sarah,

    For the vector digest I have never tried to blunt end a vector and then CAIP treat it. However normally I would cut the vector and then CAIP it immediately without cleaning it up. I have found that the buffers you use for the restriction digest work fine as buffers for the CAIP and it doesn’t seem to matter that the restriction enzymes are there. I then gel purify the vector when I have finished cutting and CAIPing it. You will lose the majority of the vector in the gel purification so I always make it the last step before ligation.

    As for the PCR clean-up I have done quite a few of these when subcloning in my PhD. After the PCR has finished you can put the whole of the PCR volume directly through a Qaigen gel extraction spin column WITHOUT doing the gel extraction. There is a protocol for doing it in the manual that the kit comes with. I then cut the cleaned PCR product with the restriction enzyme. By doing the clean-up initially without the gel extraction you don’t lose a massive amount of DNA. After the digest I then gel purify the PCR product before I ligate it. Hope this helps,

    Simon
  • Haitong Hou · Columbia University
    I totally agree with Simon Fellgett.
  • Anthony Firulli · Indiana University School of Medicine Indianapolis
    All you need to for making a vector is as follows:
    1) digest
    2) heat inactivate or phenol extract if your enzymes employed do not heat inactivate
    3) Gel check say 100ng on a gel to make sure it's all linear
    4) ETOH PPT and RS

    adding Agarose / GP chemicals to the mix can inhibit ligations.

    As for PCR just TA clone for clean up. Then you have a shuttle construct from which you can use that insert for other purposes (such as a probe as example).
  • Beston Nore · University of Sulaimani
    It is best the following, just to minimize purifications:
    Vector: Double Digest > Bluntends > Dephosphorylate > gel purify.
    Insert: PCR > restriction digest > gel purify

    The mix vector-insert, about 1:5 and 1:10 ratio for ligation
  • Yang Wu · Chinese Academy of Sciences
    you can make the last step of gel purify together in one Spin Column (the ratio of the vector-instert can be roughly judged from the gel brand ).

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