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17

Human female cells contain most of the genetic information required to make a male, but they do not contain a critical component: The Y chromosome. This is a relatively small chromosome. Wikipedia claims we have identified around 200 genes on it to date, compared to estimates of 20,000 - 25,000 genes overall in the human genome. Importantly for your ...


12

No, it doesn't seem to be common practice. A telomerase treatment would probably do more harm than good, and may be completely unnecessary in the first place. Betts and coworkers published a study in which they examined telomere length in cloned cattle. They found that while telomere length in cloned embryos does start out somewhat shorter than in ...


8

For the impatient, I'm going to say that probably if you only have a regular home freezer, its unlikely that you will be able to do this. If you have access to a -80C or liquid nitrogen storage its possible, but also less likely. Cloning an animal typically starts with the transplantation of a nucleus into an egg cell whose nucleus has been removed. So ...


8

A woman (assuming no mosaicism) has two X chromosomes in the nuclei of her cells (except for oocytes). A man, in every cell with a nucleus (except for spermatocytes), has only one, pluripotent or not. The only way he could make a female would be to either manipulate cells by duplicating the X chromosome (very difficult to do) or remove/inactivate the Y ...


7

Yes, you must fragment the genome in order to insert it into a vector for cloning; you can't "insert" the whole 5 Mbp genome of E. coli into a vector. It's difficult to transform cells with huge plasmids, 2-20 kbp is an optimal range. In any case, if you want a clone of the whole genome, wait 30 minutes and the cell will happily oblige you. Most ...


7

This answer also involves some speculations as the question is about a good theoretical framework for a science fiction. You can find in this post about how sperm can be used to produce embryonic stem cells. It would still require an oocyte for doing that. The question now is- Can you produce oocytes from a male? You may fuse two X bearing haploid ...


6

During the generation of gametes (sperm, eggs), chromosomes can cross-over - this swaps paternal and maternally-derived genetic material. So none of the descendant's chromosomes would be a direct copy of Einstein's, and furthermore, each offspring receives half of its genetic complement from each parent. This means that if you pool the DNA data from small ...


6

I have heard that Epi300 electrocompetent cells (1) from Epicentre are very efficient: > 1 x 1010 cfu/µg of pUC19. Dan Gibson used them in his paper for the synthesis of the mitochondrial genome (2). We were also thinking of using them for our assemblies, but they are pretty expensive. 1. TransforMax™ EPI300™ Electrocompetent E. coli 2. Chemical synthesis ...


6

They aren't, anymore. It was a fair guess at the time, but first I think we should define what cloning in this context mean. Our own cloning tag says: The process in nature or in the lab by which a new organism is created that is genetically identical to its predecessor. For animals - I'm going to use Dolly as an example since you do as well - when ...


6

What is the problem with handling pure mRNA nucleotides of gene X? Or even sscDNA? Problems in directly transfecting a mRNA/ssDNA mRNAs can be transfected into the cells and is quite frequently done. The problem with transfecting mRNAs directly is that the mRNA gets degraded soon and you will not have sustained expression. This is, however, very ...


5

I'll contradict the answer posted here (ages ago) and say no, it isn't possible. Growing an organ wthout MHC proteins might be achievable, but it would be rejected for not having MHC1. NK cells instruct all cells without MHC1 to perform apoptosis. In this case, MHC1 antigens are required on cells for proper self-identification. Keeping MHC1-less cells alive ...


5

Did you identify structural domains in the protein using Pfam or some other domain detecting peptide search? It's pretty useful to cut your constructs at the boundaries of structurally independent domins in the protein if you know where they are. Hydrophobicity plots might lead you to cut right in the middle of a protein fold, only exascerbating inclusion ...


5

Try dissolving at 50C. In the Qiagen gel extraction kit, it says to dissolve at 50C or until completely dissolved. The size of the gel is important too. Make sure it is 1.5% or less, and the size of the gel needs to be less than 400 mg. Also, pay attention to the first step. If the liquid is not the same color as the color of the first QG buffer then ...


5

When I optimized gel extraction in my hands, two factors turned out to be critical in maximizing gel extraction yield: The pH of the eluting solution. I used to elute the final product with DEPC-treated water initially, but the yield would vary a lot as the pH of the water changed. Using buffer EB is better, since it maintains a stable pH 8. Most ...


5

I think the best way is option #2: Suppose that your gene of interest is AAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGGGGGGGGGG and you want to insert EcoRI restriction site GAATCC Then your Fwd primer will be GAATCC AAAAAAAAAAAAAAAA and your Rev primer will be GGATTC CCCCCCCCCCCCCCCC But in general, it shouldn't matter which option you choose, as long as you ...


4

As can be inferred from the Shanks et al. paper linked in the question: In molecular biology, "suicide plasmid" is a term that refers to a plasmid which is replication incompetent.* Plasmids normally bear a sequence called "origin of replication" Ori which marks the plasmid for replication by the host cell. Plasmids that lack this Ori will not be replicated ...


4

Theoretically this should be possible as men carry both sex chromosomes. You would have to find a way to make haploid cells and then have them form a diploid cell with two X chromosomes. Your population would go through a genetic bottleneck which would soon cause a lot of other genetic problems as there is not enough variety. But besides this rather ...


4

Purification and isolation of DNA bands by cutting them from agarose gel is commonplace (Lee et al., 2012). The purification step after excision of the band gets rid of most of the EtBr and other impurities. E.g., see the websites from Isogen and Sigma-Aldrich. Reference - Lee et al. J Vis Exp (2012); 62: e3923


4

A progeny is not a half clone of its parents. Each cell expresses both maternal and paternal genes. There are no parts of the body or even a cell for that matter that is identical to one of the parents. When haploid gametes form, chromosomes are randomly segregated. So the gamete receives either paternal and maternal chromosomes (grandparents of the ...


3

Not any time soon. By far the greatest challenge is getting the DNA sequence from the computer into a form that could be implanted into an embryo. Craig Venter's team was able to build a bacterial genome from scratch, but that was incredibly painstaking work, requiring the assembly of many small fragments of DNA into a single circular chromosome. With ...


3

Just in case you are having difficulty visualising what happens in the answer from @Chris, here are the steps with the linker shown in lower case ...NNNNNGAATTCNNNNN... ds DNA ...NNNNNCTTAAGNNNNN... | V ...NNNNG AATTCNNNNN... after EcoRI digestion ...NNNNCTTAA GNNNNN... | V ...NNNNG ...


3

The recognition site for EcoRI is GAATTC, and the enzyme cuts after the first base. See this picture from NEB: The overhang is: AATT, which is supplied by your oligo and fits into the overhang. If the next nucleotide whould be a C than the site would be recreated, since its a G its not. The new sequence of the old EcoRI site is GGATTG which is not ...


3

The prototypes for these proteins are: the bacteriophage λ proteins Int and Xis, required for, respectively the integration and excision of prophages. They are, as mentioned by @WYSIWYG, recombinases. Genetic map of bacteriophage λ (source of image) the original Ihf (integrative/integration host factor) was identified as an E. coli ...


3

In my own personal lab experience, I got some unexpected results using Phusion polymerase. However, I have not seen deletions, only insertions (ranging from single bases to 3 tandem copies of a primer sequence). This is not an answer to your deletion question, but it does suggest unusual things might happen. I have seen deletions with T4 DNA polymerase ...


3

Making maps of plasmids Annotating plasmids with common features Plasmapper works through commandline Finding restriction sites Simple regex searches can do that Mapping Sanger sequencing reads BLAST will do it. Showing sequence, reverse complement of the sequence, translations in different frames EMBOSS has a collection of tools ...


3

You're essentially looking for a restriction modification system. You modify the restriction sites with methyl groups in a way that blocks restriction enzymes from accessing them. The DNA methylation should also be maintained through replication. So if you have an EcoRI site, you can use an EcoRI methylase to block the action of EcoRI.


2

The option #2 is most common. Do not forget to add 3 or more additional terminal base pairs for optimal restriction enzyme cutting (source: BioTechniques 1998, 24:582-584)


2

Disclaimer: I haven't ever done a Gibson assembly, but here is my theoretical understanding of how to design your primers. You need four 40mers each consisting of 20 bp segments derived from the vector and the insert and corresponding to the junctions that you are trying to create. In the diagram below the dotted lines represent the junctions between the two ...



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