Questions tagged [cloning]

The process in nature or in the lab by which a new organism is created that is genetically identical to its predecessor.

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Why is there no Ligase in Ligation Independent Cloning?

Ligation Independent Cloning literally says no need Ligation, but it still needs ligase to anneal the fragment to vector. And the Protocol used T4 DNA Ligase for anneal process. What does this mean &...
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#Gateway Cloning How does gene of interest replace ccdB genes?

I knew that Gateway cloning used a site-specific recombination method, but I still cannot figure out how does gene of interest actually replace ccdB genes? Bacteriophage site-specific recombination ...
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Use of plasmid pXen5 for transposon screening

I would like to use the plasmid pXen5 (by Xenogen) for a transposon screen. It contains two inverted repeat sequences, with Luciferase, Kanamycin, and the transposase itself in between. (It's tn1409). ...
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Why am I losing much more vector than insert DNA during RE cloning?

I've been cloning some inserts into a lentiviral vector (pBOB backbone) by RE digestion and blunting ends with T4 DNA polymerase. After these steps, I usually load the digested product onto 0.7% ...
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Ubiquitous overexpression line of a gene expressed higher gene expression in leaf but not in seeds

I tried to make ubiquitous overexpression lines of a gene using a constitutive promoter vector in plant. I got multiple regenerated plants through tissue cultures. At T0, T1, T2 leaves tissues, the ...
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ddT-tailed TA cloning, and the fate of a double nicked plasmid in E. coli

In an undergraduate lab class on TA cloning, it was explained that ddNTPs are used as the substrate for terminal transferase when making the T overhangs of the vector. I was told this was to ensure ...
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How can we create a living dinosaur using DNA technology?

I'm wondering what would we need to do to create a living dinosaur using DNA technology? If it is not possible with current technology, will it ever be feasible? In the movie Jurassic Park, ...
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How to avoid mutations when performing Gibson Assembly (or generating amplicons during PCR)

More often than desired (about 75 % of the time), when building plasmids via Gibson Assembly (~ 5 kb plasmids; from a maximum of 2 fragments) we obtain clones with several random point mutations all ...
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If we cloned an extinct animal, what would become of its gut biome?

If we cloned an extinct animal like the mammoth, what would become of its gut biome?
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Why does plasmid cloning efficiency go down with increased size of insert?

I am setting up a cloning experiment. Briefly, I ran PCR on a gene of interest, cloned it into a vector (pGemTeasy) and bulked it up in bacteria. I have been advised to limit the size of my fragment ...
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In traditional plant cloning, why do we require two different vectors (plasmids)?

So I was recently taught cloning in plants and I came to wonder what is the need to first put the gene of interest in the entry vector plasmid and then the final vector plasmid before finally ...
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Why is cDNA usually lacking in terminal sequences of the template mRNA?

It appears from presentations I have attended that cDNA often lacks terminal sequences (usually 5′) which have not been copied from the mRNA. This puzzles me a lot, but I have not been able to find ...
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Hierarchial and shotgun sequencing vs Massively Parallel Sequencing

Why is the cloning step unnecessary prior to sequencing with massively parallel sequencing (MPS; Illumina or similar technologies), when it is necessary for hierarchical and shotgun sequencing? I know ...
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Cloning in a large DNA fragment into a plasmid

I need to clone in a 30kb DNA fragment into my plasmid (~7kb). Working with such a large fragment I have run into a few problems. The first was purifying it from the PCR mix used to amplify it out of ...
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Is there a command line molecular cloning tool?

There are many software applications such as SnapGene, Geneious, Vector NTI, ApE, Gentle and others for assistance with cloning. Their key functions are: Making maps of plasmids Annotating plasmids ...
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Do PI-SceI and PI-PspI from NEB work in each other's buffers?

I'm considering a plasmid design strategy that would allow me to use restriction enzymes to exchange plasmid inserts among different backbones. In this case, the plasmids would first be made through ...
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Can someone explain DNA methylation reprogramming in simple terms to me?

From what I understand, one of the challenges of cloning is that the DNA methylation that is supposed to occur during embryonic development does not occur normally and this prevents natural gene ...
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How to DIY preserve pet DNA today so that it can be used in 20 years

I know that there are companies that offer a kit+storage of your pet DNA for around $2k. My question is if there is any other option for doing this yourself with a thought in mind that in 20+ years ...
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"Antibiotic resistance" equivalent in archaea for selection during cloning

I'm beginning to work with halophilic archaea and I'm trying to figure out a good way to select for cells that have taken up a plasmid. Obviously, one can't use antibiotic resistance since they are ...
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Using PCR to add the overhangs to gBlocks for NEB HiFi

Does adding the overhangs to gBlocks for NEB Hifi using PCR pose a big problem? The IDT website suggests that one should design the gBlocks with the overhangs. However, if I did not do this, will it ...
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Sansevieria falling back to the wild form via leaf cuttings but not via micropropagation

I have heard that when you propagate Sansevieria via leaf cuttings, you get the wild form again. But if you use only meristem tissue, you can propagate the multicolored cultivated forms. What would be ...
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Ligation without purifying insert

I am planning to insert a 45 bp sequence in a vector. After restricting my insert to create compatible sticky ends, I am finding no way to clean it up. Is there any way for cleaning this fragment ...
3 votes
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T-vector creation

I'm working on TA cloning for the first time and I need to turn some existing plasmid backbones into T-vectors. I'm using a Klenow fragment (3' -> 5' exo-) to dA-tail my inserts. Is there any ...
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Production of plant derivatives using genetic engineered micro-organisms

I saw a Thought Emporium video where spider silk was produced by genetically modifying yeast. I have also read about companies making vanillin (vanilla flavour) using this technique. I am curious to ...
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Plating 96-well bacterial transformations

I need to clone 96 different inserts into the same backbone in arrayed format. I am planning to perform Gibson assembly reactions in a 96 well plate, and then do chemical transformations in a 96 well ...
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What if target DNA doesn’t have restriction sites

All the examples on DNA cloning I have encountered have assumed that the target gene and vector both have compatible restriction sites at just the right locations (probably for ease of explanation). ...
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Calcium ions and bacterial transformation

What is the mechanism by which calcium ions and heat treatment allow the bacterial membrane to become permeable, allowing the uptake of plasmids?
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Alkaline Phosphatase and Ligase Protocol for Cloning

In the image the circular molecule is a restricted vector and the linear red molecule is a DNA insert. I found this protocol in my lessons notes, but I don't understand how it is possible that ligase ...
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Will this strategy work for verifying successful cloning of a DNA fragment into a plasmid vector?

So I am in a bit of a time constraint. Essentially, I inserted a DNA fragment via molecular cloning which contains a unique RE site. I need to confirm whether my colony has or does not have the ...
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Can Mung Bean Nuclease only digest a ssDNA 5' overhang?

Can Mung Bean Nuclease only digest a ssDNA 5' overhang, if the double-stranded region of DNA (20bp) is A-T rich at the ends? Does this enzyme degrade 5' overhang (5bp or 30 bp) with 100% efficiency?
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Cloning using pET28a and Protein Expression in DH5alpha and BL21

Can someone please direct me to an e-resource or a book that will help a newbie like me learn in depth about Cloning using pET28a and Protein Expression in DH5alpha and BL21. Though I have done ...
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Why would a mammalian vector plasmid require an antibacterial resistance gene?

I have been trying to understand the composition of plasmids used in recombinant DNA cloning, such as this: https://www.genecopoeia.com/wp-content/uploads/2020/02/pReceiver-M35-051818.pdf and I am ...
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Blood and hair follicles from 25 people put on the Moon; enough to regenerate a human population?

Discussion on the question Why were blood and hair follicles from 25 people put on the Moon? calls into question whether this is a stunt or might have any basis in science. There's two parts to my ...
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Why we need an egg cell donor and a surrogate mother in an organism cloning process?

Could a whole set of artificial organism-cloning process only be operated on the somatic nuclear donor ,which means for example ,can we extract the somatic nuclear from animal "A" and put it into the ...
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Protecting an internal clevage site from restriction enzyme while cloning

2 restriction enzymes used to generate cohesive ends in a vector and foreign DNA(gene of interest you want to clone) also cuts the foreign DNA at an internal site. What are ways to overcome this?
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Is SCNT possible with only the nucleus of a somatic cell (rather than a whole somatic cell)?

The following figure from Life: The Science of Biology (11th edition) explains how SCNT (somatic cell nuclear transfer) was used in the cloning of Dolly: (The original paper about cloning Dolly - ...
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If a woman was fertilized with her own DNA, would it be her clone?

Several questions: 1) Can human DNA fertilize an egg, without having been naturally packaged in a sperm cell? 2) If a female had her ovum fertilized with her own DNA, would the fetus be her clone? ...
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What is the use of having a surrogate mother in animal cloning?

In animal cloning, an ovum is collected by an animal and processed to form an embryo after fusion, which is going to be inserted to a surrogate mother. So my question is that why can't the embryo be ...
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Unusual terminology of vector used

What is the significance of a+ in the name pET-28 a(+)? Is there any a- strain and differences between a,a+,a-?
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Why does one combine PCR and cloning as ways for amplification of sequences?

Why does one combine PCR and cloning as ways for amplification of sequences? Don't they produce the same result? I was reading the paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864885/ and got ...
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What does it mean for a gametocyte to "reprogram" the genes inside of it?

I am aware that frequently clones have genetic defects not present in the donor organism, even though the two are genetically identical. The reason for this is that apparently the enclosing gametocyte ...
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If bacterial plasmid DNA is methylated then how do we digest it using restriction enzyme in cloning experiments? [duplicate]

So, I was reading about how bacteria protects its own DNA by methylation against restriction enzyme. But if the plasmid DNA is also methylated then we can't cut plasmid using restriction enzyme for ...
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What is the sequence of pPROLar.A plasmid?

I've been looking for the sequence of pPROLar.A122 plasmid but I haven't found it. Where could I find the sequence? The first link (addgene) on googling reports a plasmid which is reported to have ...
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If bacteria with recombinant DNA molecules do not survive in gene cloning, how they can be grown into a colony after being isolated?

This is as far as my understanding of gene cloning goes. Let's say that the plasimd (vector) in the bacteria contains an ampicillin resistence gene. Through restriction enzymes, the ampicillin ...
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How can seedless fruits not be GMO?

Biology is the closest I could find to botany on SE. Grafting can not occur naturally and so if a fruit does not have seeds there is no way it could reproduce in nature. That logic makes complete ...
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Why is DNA polymerase added at the end of PCR reaction?

PCR reaction is used to amplify DNA fragment. Each reaction requires a DNA template, buffer, dNTP mix and a unique pair of primers, one ''forward'' primer and one ''reverse'' primer. The DNA ...
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use of antibiotics in selection of good plasmids

I have not understood the role of antibiotic medium in filtering the bacteria containing the recombinant plasmids with the ones having normal plasmids. Since all the plasmids of a certain bacterial ...
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Are clones less likely to be fertile?

I know that hybrid animals like mules are infertile. What about clones? Do cloned animals show the same fertility as their original?
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Thousands of Reporters Integrated in Parallel (TRIP) barcodes

Has anyone worked with constructing barcodes for TRIP? How does constructing of barcodes work in general? I don't seem to be able to grasp the basics of constructing these barcodes.
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Misuse of DNA leaks to create your clones, or children

Recently, sequenced DNA data leaks have been reported. The linked article discusses possible risks like blackmailing based on health related data. However there could be one more misuse, at least ...