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Prof. Allen Gathman has a great 10-minutes video on Youtube, explaining the reaction of adding nucleotide in the 5' to 3' direction, and why it doesn't work the other way. Briefly, the energy for the formation of the phosphodiester bond comes from the dNTP, which has to be added. dNTP is a nucleotide which has two additional phosphates attached to its 5' ...

DNA replications needs a source of energy to proceed, this energy is gained by cleaving the 5'-triphosphate of the nucleotide that is added to the existing DNA chain. Any alternative polymerase mechanism needs to account for the source of the energy required for adding a nucleotide. The simplest way one can imagine to perform reverse 3'-5' polymerization ...

Unlike in an SDS-PAGE, where the SDS adds negative charges to all proteins, in a standard agarose gel for nucleic acids all proteins should move according to their own charge. You can get a rough idea in which direction Taq would move by getting the amino acid sequence and calculating the isoelectric point at the pH you are running your get at. It might be ...

According to their website New England Biolabs use a version of the approach pioneered by Wayne Barnes, as described in: Kermekchiev, M.B., Tzekov, A and Barnes, W.M. (2003) Nucl. Acids Res. 31, 6139–6147 This is basically an assay for the mutation rate in a PCR-amplified lacZ (β-galactosidase) gene, assayed by transforming E. coli, plating on the ...

You can look up Gibson Assembly or Circular Polymerase Extension cloning (CPEC). For both of these the website for J5 has some good protocols. Here is the one for CPEC: http://j5.jbei.org/j5manual/pages/80.html For CPEC you can look at the 2011 Quan paper: http://www.ncbi.nlm.nih.gov/pubmed/21293463 Hopefully that helps.

it is highly variable even in a single cell type. Dont know for a human cell but this is the range for ecoli: 1500-11400 check this site out. got this number from there (its quite useful for questions like these): http://bionumbers.hms.harvard.edu/bionumber.aspx?&id=101440&ver=3&trm=RNA%20polymerase You can estimate protein copy numbers and ...