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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196 views

Organ cloning - possible to make a non-antigenic organ?

From a J. Neil Schulman article on Organ Cloning: Cannibalizing organs from other people also entails the risk of rejection because of incompatibilities, not only for tissue-typing but also ...
12
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3answers
2k views

Does a man contain all the genes needed to make a woman?

This question is brought on by a Sci Fi novel I am thinking about writing. The plot device involves a colonist in charge of building a population on a new planet who loses his supply of embryos and so ...
11
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1answer
4k views

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 ...
11
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1answer
343 views

Deletion errors with Phusion Polymerase?

I was wondering if anyone has ever seen large deletions (~40-50 bp) occur when using phusion polymerase to clone a gene. I have recently tried using Circular Polymerase Extension cloning to clone a ...
10
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2answers
697 views

What is the highest competency possible for E coli?

I am looking to find a highly competent E coli strain. I am making a library of a ~6.6kb plasmid and I am not getting high enough efficiency. Does anyone have a suggestion of a strain/protocol with ...
10
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1answer
690 views

Cloning and Telomeres

I read that the length of telomeres in Dolly was shorter, because the DNA was taken from an old sheep. Shorter telomeres may lead to early aging. What is happening now with cloning? Do we use a ...
8
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2answers
266 views

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 (...
7
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2answers
1k views

How do I prepare and clone from E. coli DNA?

I'm looking for a protocol to get genomic DNA from an E. coli sample so that I can clone a small portion of it using PCR into a plasmid. (< 500 bp in this case). It seems OWW (Open Wet Ware) ...
7
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1answer
432 views

Protein construct design

I am trying to create some constructs of a certain protein deleting well defined domains (at either terminus) to determine interaction regions with other proteins etc., 3 constructs with varying start/...
6
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1answer
996 views

Does anyone have any TOPO directional cloning tips? [closed]

I'm just about to start working on a TOPO cloning after I couldn't get it to work with standard restriction/ligation. Does anyone have any tips for TOPO cloning?
6
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1answer
307 views

How bad is ethidium bromide in your plasmid for downstream applications?

I want to transfect Adipose Derived stem cells using a clone of my own. My problem is that I get a lot of contamination (not only genomic, but due to the nature of the technique, I get undesired ...
5
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2answers
3k views

Primer design for introduction of restriction sites flanking a gene of interest

I am wondering what the correct method for primer design to introduce restriction sites. Specifically between two methods. 1) Primer first partially hybridises to the gene, has a mis-match where the ...
5
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1answer
351 views

Why are female clones more often produced

As a student of biology when ever I come by artificial cloning, I always find examples of females being cloned - Dolly the sheep, CopyCat, Daisy, etc. The only male I could see was Fibro mouse and a ...
4
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2answers
110 views

Is there a command line molecular cloning tool?

There are many softwares such as SnapGene, Geneious, Vector NTI, ApE, Gentle and others for assistance with cloning. Their key functions are: Making maps of plasmids Annotating plasmids with common ...
4
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1answer
20 views

Why does the cloned cat, CC, exhibit mosaic fur color?

Since fur color in this cat is from a sex-dependent gene, I see how calico cats occur. However, I don't fully understand why it happens in cloned cats. When the cat is cloned, the DNA is taken from a ...
4
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1answer
171 views

Freezing after restriction digest

Brief question: I have performed a double digest with heat inactivated restriction enzymes with no star activity. I usually purify the DNA (PCR inserts and linearised vector) using a kit or gel ...
3
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1answer
2k views

Does a woman contain all the genes needed to make a man?

I know the answer of inverse is at here, but how about this question? I also read this question, can I imply to human that a woman can also contain all the genes needed to make a man? Edit: I ...
3
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1answer
962 views

How much DNA of Albert Einstein is recoverable?

Since there seem to be five biological descendants of Albert Einstein, and the original chromosomes of him distributed among them ... Is it possible to recover enough DNA of an individual from his ...
3
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1answer
9k views

What is a suicide plasmid?

What is a suicide plasmid? Example: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737453/ mentions a "suicide vector". See also http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/allele-...
3
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1answer
256 views

Is the amount of dNTPs rate limiting for very long PCR products?

I'm using the Q5 system and I'm PCRing a product that will have a final length of around 9500 bases. I have noticed that the product is there but very faint. The primer seems to be used up. So my ...
3
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1answer
74 views

Is it possible to clone a Neanderthal?

As Neanderthal DNA has been sequenced, is it possible to create a living Neanderthal?
3
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2answers
302 views

Neomycin resistance gene in E. coli stb3 cells

We use a lentivirus packaging vector called pLenti_puro_DEST. We use puromycin to select for the cloned gene in 293T cells. However, the pLenti_Neo_Dest has been discontinued, so I'm cloning a ...
3
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1answer
61 views

Literature about putative epigenetic state changes in mammal sequences after cloning steps in Escherichia coli

I would like you to point me out some literature about putative epigenetic state changes in mouse/mammal sequences after cloning steps in Escherichia coli. This are the last search details I used in ...
3
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1answer
719 views

Open protocol for Ligase Independent Cloning

Ligase Independent Cloning is a protocol that allow an insert to be integrated into a vector without ligation. It uses T4 DNA polymerase with only ATP to first chew back from blunt ends to create ...
3
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0answers
63 views

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 (...
3
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0answers
31 views

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'...
2
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3answers
2k views

Improving Gel Extraction yields

How can I improve my Gel Extraction yields. We use the standard protocol from Qiagen, gel extraction, dissolve in QG buffer at 42C and purify via anion exchange columns. However, with 500 ng we ...
2
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2answers
222 views

In Sanger sequencing, why do we resort to cloning? Why doesn't PCR suffice?

I understand that in Sanger sequencing we can clone our fragments with the help of e.g. bacteria to make multiple copies of our fragments for further analysis. I also understand cloning can be a ...
2
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1answer
191 views

What are the Gateway clonase enzymes?

The Gateway cloning system utilizes what Life Technologies refers to as "Clonase enzyme mix" to catalyze the BP and LR reactions. What is in this enzyme mix? Is there a sequence for them?
2
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2answers
2k views

Primer design for Gibson assembly

I'm trying to design a primer for Gibson assembly. My gene of interest is on a plasmid, and I want to copy that gene, and put it into a different plasmid. I am unsure how to design my primers for ...
2
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2answers
279 views

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, ...
2
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1answer
65 views

Cloning a gene of an organism with an nonsequenced genome

What would be the best protocol to clone a gene about 5kb in size? The genome is not sequenced, but the gene itself very similar to orthologous genes of organisms with known sequences.
2
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1answer
44 views

Cloning Ice Age Lion Cubs

It was just on the news that scientists discovered "Two cubs in Russia's Sakha Republic last August in a near-perfect state". And the South Koreans and Russians are attempting to clone these extinct ...
2
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0answers
32 views

“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 ...
2
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0answers
51 views

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 ...
2
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0answers
313 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 ...
1
vote
1answer
55 views

What are the problems in using an ssDNA or an mRNA for expression instead of using a plasmid?

Let's say you want to do a gene cloning experiment. You have an empty expression vector (mRNA or DNA) and are looking to clone some gene X into it. Why is it you can only buy X already inside a vector ...
1
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2answers
321 views

Removing a restriction site and introducing other at its place

What would you do if you want to remove an EcoRI restriction site and introduce BamHI restriction site at apprx. the same location ? One of the answers in my textbook was : To construct a DNA ...
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2answers
73 views

Alternatives to His-tags

What are the alternatives to using a histidine-tag in detecting protein-protein interactions? Why would they be more beneficial than his-tags?
1
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2answers
1k views

Why doesn't T5 exonuclease have endonuclease activity in Gibson assembly?

Gibson assembly uses T5 exonuclease to chew back the 5' end of dsDNA to generate overhangs. However, T5 exonuclease, in contrast to lambda exonuclease, is reported to have ssDNA endonuclease activity (...
1
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1answer
21 views

Unwanted E. coli product in plasmid product

Using a TOPO XL Cloning kit, our lab took some amplified human DNA, inserted it into the vector, and attempted to clone it in E. coli. However, upon sequencing the purified plasmid product, the ...
1
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1answer
22 views

Methods for varying insert size in E. coli clones

I'm writing my master's thesis proposal, and I need to improve the solution-behavior of some truncated protein domains which have previously shown poor solubility when expressed in E. coli. To ...
1
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1answer
27 views

What's the rationale behind freezing one's eggs if cells can be cloned?

It seems to me there's a lot of discussions around the topic of somebody freezing her eggs. What I don't understand though are the following: Why would you freeze your eggs when you know that the ...
1
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1answer
23 views

Using PGAL10 promoter to overexpress specific yeast genes: How to?

I was reading a poster about expression boosting for Artemesinin production in yeast and it had this line: PGAL1 and PGAL10 have been used for the overexpression of all genes from the ...
1
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1answer
160 views

Does DNA Ligase look for complementarity in sticky ends?

Can DNA ligase seal two non-complementary sticky ends if the reaction is incubated at the optimum conditions ? I do almost 100% double digestion in the lab. However, during ligation, when I do a ...
1
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1answer
158 views

Molecular Cloning- Blunt end restriction endonucleases

I work in a microbiology lab where we do a lot of cloning. I have always used restriction endonucleases to cleave the DNA to have sticky ends and not blunt ends. I currently am working on a project ...
1
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1answer
140 views

Possibility of Human cloning

Technology has evolved so much as to clone animals. But, what about human cloning? Is it really possible to create humans in lab? Or is it that technology has not advanced to that level..? Why cant ...
1
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1answer
48 views

How can I learn about how cloning works in nature (twins)?

I would like to learn about how the process of cloning works in nature - how are twins "created" after fertilization what genetic changes can occur in each of the twins. Where should I start? What ...
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0answers
25 views

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....
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0answers
34 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 ...