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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Commercial suppliers of cloned vectors?

I am a recent transplant from academia to industry. I would like to follow-up on some experiments in the literature, for which I need cloned plasmids. Addgene of course doesn't (for the most part) ...
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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 do Gibson in 96 well plate, then do chemical transformations in 96 well format. However, I am not sure ...
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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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Pet 28a plasmid is obtained from which type of bacterial strain

I want to ask some question which is very confusing Pet28a is a vector.I want to know which strain of bacteria have pet28a plasmid? E.coli DH5 alpha strain have pet28a plasmid? Please help me .......
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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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What factors make iSCNT less effective than SCNT?

When comparing the efficiency of SCNT vs iSCNT, specifically what percent of transfers that generate a living animal; it appears that genetic distance between the receiving cytoplasm and the donor ...
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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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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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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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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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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 ...
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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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668 views

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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Insertion Confirmation Cloning Strategy

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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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 ...
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Are cloned spieces significantly more vulnereble to deseases than sexually reproducing species?

I would like to be able to compare the risk for species to go extinct implied by their reproduction mechanism in the very short term. Imagine we choose some species A that can reproduce both sexually ...
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210 views

Is it Theoretically Possible to Create a Male clone from the donor Cell of a Female?

I understand that Dolly the Sheep was created without the presence of a sperm cell. Instead, nucleus DNA from somatic cells of adult sheep were used. Since Dolly was female, i am assuming that the ...
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Which comes first, PCR or Gel electrophoresis?

If I want to find out wither a group of patients have the abnormal BCR-ABL cancer gene, how do I benefit from the PCR and the gel electrophoresis techniques? I'm kinda lost trying to determine which ...
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Are drones from one honey bee queen all clones?

The Honey Bee queen lays eggs that develop into worker bees (female) if she fertilizes the egg. If she does not fertilize the egg, it will develop into a drone (male). The drone only has the queen'...
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Why is easy to clone cats but it's hard to clone dogs?

According to this article, Here is why we're not cloning humans there are species who are easy to clone and other who are hard to clone. Cats and mices would be in the easy to clone set , and dogs and ...
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Isolating gene of interest from a large strand of DNA [closed]

I was always confused about this in class and never learned it fully. So say you have 10,000 bp of DNA and you just want to isolate 100bp of specific region(gene of interest) using restriction enzyme. ...
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Can you Interrupt a promoter region by inserting another promoter region into it?

I currently have a plasmid (pFPV25.1) which contains a promoter region with multiple cloning sights in it. I would like to place a new promoter I have isolated, and I was wondering whether I could ...
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Cloning problem. Digestion test at the end of the cloning is always negative

I have a problem with my cloning, I'm trying to clone a 483 pb in a pSecTag vector with restriction sites HindIII and BamHI. Here are the procedures I'm doing: • I introduce the restriction sites ...
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Somatic cell cloning always produces female offspring? [duplicate]

I was looking at the somatic cell cloning of goats. One of the advantages of the process said was All offspring produced are female. Now I'm confused. How is this the case? I know that the ...
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Cloning DNA fragment - at least trying to

I`ve been trying to clone a fragment (about 1.6 kb) using pGEM system, unfortunately it was not succesful. Then, I moved to pJET, and guess what? Again, very low transformation efficiency. Now I am ...
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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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Plasmid transformation yields only empty vectors

I have to electroporate pMAD cloned with an insert of 7.4 kb in my S. aureus isolate. When I electroporated in RN4220 I had no problems but when I put it in my isolate I just recover few colonies and ...
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338 views

LacZ' selection: blue colonies despite ligation of insert

It has been suggested that bacteria transformed with pBlueScript vector, containing an insert in the middle of the lacZ' gene, can still give blue colour on X-gal, if the insert is small and ligated ...
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351 views

What step of the cloning process requires an egg cell? Have alternatives been attempted?

In the famous Dolly the sheep an egg cell was required. Why was specifically an egg cell required compared to say sperm cells or any other cell types? Given that egg cells are not trivially harvested ...
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Genetic information storage and the extent to which it controls appearance/structure

So there are often articles about cloning from a small piece of tissue etc. My question is (two actually) Is the Complete information about entire organism's structure stored (as in duplicated) all ...
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Complementation cloning: what, why and how? [closed]

I get that you're cloning a whole cDNA library, but why does E. coli need a complementary gene to allow the gene expression? Why is that complementary gene considered to be a mutant? AND how does ...
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Can a human be made with ovum but without sperm?

This article says that scientists were successful in making a mouse using only a sperm and no egg (ovum). The article also states that this procedure could be applicable to humans. However, I want to ...
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Difference between clonal and subclonal mutations

I'm a physicist writing a proposal that has to do with cancer as a disease driven by evolutionary selection. As far as I understand, all tumors start with a single precursor (single cell or group of ...
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Why doesn’t p53 cause the repair of cellular DNA that has been altered experimentally?

When scientists mutate bacteria or even embryos of lambs and other animals, why doesn't the p53 reverse whatever mutation the scientists cause? I know that the p53 stops the DNA from mutating and ...
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When is strict sterile technique necessary? Cloning vs. protein expression

Background: I have previously worked with RNA, and then we use laminar flow hoods for any work where we do plating or inoculation of cultures while applying sterile technique. I am now working in a ...
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Maximum loading volume for agarose gel with 10 well comb

I want to load my agarose gel with my PCR products, which are 50 µL reactions. I am wondering how much of those reaction I can load into one well if I use a standard agarose mini gel (10 well comb, ~...
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Can you make sperm and eggs from the same person using in vitro gametogenesis?

In a recent popular science article there is a discussion of the potential disruptive technologies that could become available with in vitro gametogenesis (IVG). Using skin cells you will be able to ...
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Is it possible to both extract DNA sequences from plasmids and fuse them using primers for Gibson assembly?

Maybe I'm overcomplicating this, but I'm having some issues with a construct. So, I have three plasmids containing a total of eight DNA sequences that I need to fuse into one linear sequence. I plan ...
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Is a Linker sequence mandatory in primers for PCR Cloning?

I have designed the primers for PCR cloning, but i was not getting any colonies after the ligation. One of our colleagues said, that the restriction digestion might not be working as there is no ...
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What is the typical amount of full length clones in a cDNA library?

For a Yeast Two Hybrid project we need to make a cDNA library from a single celled organism. We used the Cloneminer II kit from Thermo. We followed the manual and performed all the quality control ...
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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 ...