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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1answer
255 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 ...
3
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2answers
50 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 ...
2
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1answer
58 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 ...
2
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2answers
107 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, ...
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0answers
43 views

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 ...
1
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1answer
59 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 ...
2
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0answers
38 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 ...
9
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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 ...
10
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1answer
324 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 ...
4
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2answers
60 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 ...
1
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1answer
81 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 ...
3
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0answers
26 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: ...
1
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1answer
95 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 ...
2
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0answers
28 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 ...
3
votes
1answer
442 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
votes
1answer
4k 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 ...
2
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1answer
94 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
votes
1answer
54 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.
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2answers
122 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 ...
1
vote
2answers
516 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 ...
0
votes
1answer
31 views

What happens between making a DNA library and screening by nucleic acid hybridization?

I understand how a plasmid library is made and how each clone is put on the nitrocellulose membrane and nucleic acid hybridization is done. What I don't understand is how each clone is "identified" ...
0
votes
2answers
66 views

Telomeres in cloning experiments

In whole animal cloning ( eg. Dolly ) the nucleus is taken from a somatic cell. So isn't shortening of telomeres a problem ?
3
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1answer
63 views

Is it possible to clone a Neanderthal?

As Neanderthal DNA has been sequenced, is it possible to create a living Neanderthal?
2
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0answers
218 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
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1answer
44 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 ...
2
votes
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
votes
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 ...
5
votes
2answers
141 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 ...
-3
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1answer
148 views

How much can be said about behaviour (future) based on bloodgroup?

My bloodgroup for example is A+. How much information is knowing my bloodgroup? How much information can be known from a blood sample? I expect you to be able to clone if known a complete DNA sequence ...
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
votes
1answer
636 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 ...
11
votes
1answer
313 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
votes
1answer
2k 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 ...
7
votes
1answer
246 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 ...
6
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1answer
833 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?
5
votes
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 ...
10
votes
2answers
508 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 ...
13
votes
1answer
180 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 ...
7
votes
2answers
967 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) ...