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There are stable ubiquitinated proteins in mammalian cell lysates even if active proteosomes exist in cells. First, you might want to make sure that the antibody is applicable to WB. Then, you would ask if your WB system using the antibody works. You could optimize the condition using just 1D SDS-PAGE followed by WB. For the condition for isoelectric ...


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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


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In fact, it is rare that stop codon mutation causes translation of polyA sequence in mammalian cells because you will find another in-frame stop codon downstream. And some stop codon mutations still producing the protein stably(1). However, as WYSIWYG mentions, in the absence of alternative in-frame stop codon, mRNAs would be degraded. It is also called ...


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Yes you are correct. These mRNAs, that lack stop codon will cause translation to continue into the poly-A tail (it will result in addition of lysines not phenylalanine). Since no stop codon is present, the ribosome remains attached to the mRNA. Under these circumstances, a pathway known as non-stop decay is activated. An important protein in this pathway — ...


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No, this will not happen. mRNAs are inspected in the nucleus before they are exported into the cytoplasm (at least in eukaryotes), where transcription and translation don't happen at the same place. This ensures that no mRNAs without stop codons or premature stop codons are exported. This phenomenon is called "mRNA surveillance". mRNAs that do not pass this ...


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This is more of a hypothesis I am not sure if suffocation per se would cause pain. Asphyxia, as RoryM indicated in their comments, can lead to anxiety and panic but not really pain. However, forceful breathing may lead to muscular fatigue which may result in pain. Pain induced by muscle fatigue is called myalgia. Myalgia is possibly triggered by low pH ...


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Most restriction enzymes have a 6bp restriction site (some have 8bp site also). So two restriction sites generally have to span 12bp. However some restriction sites can overlap. For example BamHI and SmaI: BamHI |______| GGATCCCGGG |______| SmaI In this case the total length has reduced to 10bp. However such combinations are not that common and ...


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Mutations in DNA or RNA sequences do not necessarily result in significant changes in the functions of the proteins they encode (or in the case of RNA ribozymes, ribozyme function ). This is because, for a variety of reasons, the change in DNA/RNA sequence may not significantly alter the structure and function of the ribozyme/protein (the function that ...


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Just expanding on aandreev's comment. Polyadenylation is initiated by binding of CPSF (Cleavage and Polyadenylation specificity factor) after its binding to the AAUAAA motif. From the introduction of this article: CPSF (cleavage and polyadenylation specificityfactor) binds to the AAUAAA hexamer via its 160-kDa subunit and possibly its 30-kDa subunit ...


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In this paper (Li and Elledge, Nature Genetics, 2005) it is shown that the PGK promoter works in E.coli but it has very little activity. So, I don't think it is suited to properly express resistance enzyme. About Neomycin, many aminoglycosides resistance enzymes (not all) that work on Kanamycin works well also with Neomycin. I have personally selected ...


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PGK is mammalian (murine) phosphoglycerate kinase promoter. It will therefore not work in bacterial systems. Why don't you select using ampicillin only? You can increase the ampicillin concentration if you want. Regarding: I can't find anyone selecting bacteria by Neomycin anywhere. Kanamycin is generally used for bacterial selection. The Neomycin ...


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It's because E.coli BL21(DE3) are competent cells. The competent is the key here as the cells were chemically treated so the transfection can be performed by heat-shock with high efficiency. This means these bacteria are quite fragile, due to the chemical treatment, and therefore are very sensitive to both mechanical and thermal shocks. Pre-heating the ...


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Tryptone is hydrolysate of casein- a milk protein- and may contain residual lactose as impurity. It is one of the constituent LB media. Addition of glucose may prevent induction of the cloned gene under lac promoter (or T7 polymerase under lac promoter in case of T7 promoter vectors) by catabolic repression. Induction of protein production is not desirable. ...


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From the comment section: Mostly segregation of PCR-amplified DNA from non-amplified DNA. Typically no DNA or sample should ever enter a specific room designed to prepare the MasterMix for PCR (this is true for diagnostic labs, in the research one I never saw that). For the rest, following good laboratory practices (google that) should be enough.


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Well the simplest way is to cut the gene at the both ends from operon by means of proper restriction enzymes but in case that you get more than one recognition site then you can use microRNA or proper oligo to make a hairpin loop and break that part If you only want to silence the operon then you just need to cut it nearly at middle of the gene and ligate ...


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In any PCR reaction ( including the assembly PCR ) the substrate of the reaction is a mix of mono nucleotides. Specific oligos serve as primer by annealing on the template and that are extended by a DNA polymerase. It is a polymerization reaction. In the LCR there are no mono nucleotides instead you have many different oligos that anneal one after the other ...


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In short, electrostatic forces and mechanical forces. Electrostatic forces are responsible for the classic Watson and Crick base pairing that is de facto what brings together codons and anticodons. Mechanical forces are involved in the displacement of the pairing and the shift of the RNAs. The ribosome's subunits literally grab and move both messenger and ...


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It's actually very simple. You misunderstood the term polymorph in this situation. A microsatellite is a sequence composed of a short repeated DNA motif. A polymorph satellite is then simply a microsatellite varying in length between individuals, i.e. composed of more or less repeats of the motif. The flanks of the microsatellite are the DNA sequences ...



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