Tagged Questions

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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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
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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: ...
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0answers
40 views

Possibility of Human cloning

Techonology 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 ...
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0answers
19 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
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1answer
191 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 ...
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0answers
58 views

How can I get higher concentrations of PCR products

I am trying to multi-fragment Gibson Assemblies and my PCR product concentrations are not high enough to yield plasmids every time. I am using a very low amount of elution buffer to maximise ...
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1answer
1k 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 ...
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0answers
32 views

Cloning an extinct species - how far are we? [duplicate]

What is the state of the art regarding cloning an extinct species? I don't mean Jurassic Park stuff, but for example extinct animals for which well preserved DNA and a reasonably adequate extant ...
2
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1answer
46 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?
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34 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
55 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
272 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 ...
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1answer
28 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" ...
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2answers
60 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 ?
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1answer
46 views

Is it possible to clone a Neanderthal?

As Neanderthal DNA has been sequenced, is it possible to create a living Neanderthal?
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0answers
144 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
37 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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3answers
950 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 ...
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0answers
46 views

Experience with Detox Cloning

I just saw this paper. A Highly Efficient Molecular Cloning Platform that Utilises a Small Bacterial Toxin Gene I was curious if anyone had experience with this cloning strategy using the IbsC ...
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3answers
1k 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 ...
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0answers
78 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 ...
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1answer
116 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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0answers
47 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
550 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 ...
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1answer
276 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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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
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1answer
198 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
723 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?
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2answers
2k 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
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2answers
346 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 ...
9
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1answer
111 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 ...
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2answers
781 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) ...