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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How does LCR compare to Assembly PCR

The question pretty much explains itself. How do the two methods compare? I've always used Assembly PCR but the method is prone to mistakes and I'm curious how it compares to Ligase Chain Reaction (...
bobthejoe's user avatar
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How Many Generation We can Run a Mycelium of Pleurotus ostreatus/Pearl Oyster?

I am able to grow mycelium successfully on the spent coffee grounds. And mycelium is successfully fruiting once/twice on the same substrate. I am cloning by taking mycelium from the existing one (...
Amol M Kulkarni's user avatar
3 votes
1 answer
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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). ...
Julius's user avatar
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Gibson assembly - primer design with A and T rich regions

I have question about Gibson assembly. I have done it several times and it always worked okay for us, but now I want to assemble together a fragment which has sequence like this: 5'...
MartinK's user avatar
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2 votes
1 answer
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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% ...
Mary's user avatar
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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 ...
yukashima huksay's user avatar
2 votes
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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 ...
A. Radek Martinez's user avatar
2 votes
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What should be the distance between plant promoter and gene?

I am trying to clone a rice gene under a different endogenous rice promoter.I will be cloning the CDS of the rice gene.So I wanted to know what is the minimum or maximum distance I should put between ...
astroboy89's user avatar
2 votes
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460 views

Can Pfx polymerase add only one 3' A overhang?

I am trying to clone a PCR product that was amplified using Pfx polymerase into pGemT vector. I had to A-tail the PCR product using Taq polymerase since Pfx only generates blunt end products. My ...
pepe84's user avatar
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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 ...
Gordon Chao's user avatar
1 vote
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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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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?
plaza-moon's user avatar
1 vote
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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?
user110134's user avatar
1 vote
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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 - ...
Oren Milman's user avatar
1 vote
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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 ...
Marina's user avatar
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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 ...
CDB's user avatar
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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 ...
mimat's user avatar
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How to specify a promoter in de novo gene synthesis service?

I am trying to use a de novo gene synthesis service (Genscript) and one part confuses me: Where do I pick the choice of promoter? Or is the promoter choice automatic based on my plasmid choice? e.g....
curious_cat's user avatar
1 vote
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275 views

UV light-induced mutagenesis during gel extraction

this is a very short question I did not find the answer for online, neither on this nor other fora. At the beginning of my cloning protocol, I extracted the band with the sequence of interest directly ...
pat_krat's user avatar
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de novo gene synthesis vs cDNA library creation from plant RNA: Pros & Cons

What are the relative pros & cons of the following two approaches when one wants to insert a known, sequenced plant gene into a target host organism: de novo gene synthesis vs plant matter ...
curious_cat's user avatar
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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 &...
Gordon Chao's user avatar
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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 ...
bio's user avatar
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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 ...
Danyn Patel's user avatar
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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 ...
Ross Jones's user avatar
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222 views

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 ...
Carica Rubus's user avatar
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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 ...
Imprisoned Rhesus's user avatar
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53 views

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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What tools are used in animal cloning?

So I was reading up on articles to do a project about cloning but there were no places in the article where It states the tool used to take the gene out of the nucleus and insert it into plasmids ? ...
random user's user avatar
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Closing a Plasmid by Ligation

I have been having trouble with what should be a fairly simple ligation. I had a plasmid that I needed to cut part out of, so I designed a couple PCR primers to do that. The plasmid already had a ...
user137's user avatar
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codon optimization / enzyme active site improvement

Does one necessarily have to use de novo synthesis of DNA when attempting protein expression improvement by codon optimization? Or are there other ways? e.g. Say the gene coding for the enzyme needed ...
curious_cat's user avatar
-1 votes
1 answer
47 views

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-?
Arnab Ray's user avatar