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8

I think this is due to the over-representation of recognition sites with length 6: data<-c(16, 16, 12, 12, 6, 6, 6, 6, 4, 16, 6, 6, 6, 6, 15, 15, 6, 6, 6, 6, 11, 11, 6, 6, 4, 4, 6, 6, 11, 12, 6, 6, 23, 23, 6, 6, 6, 6, 9, 12, 4, 4, 6, 6, 6, 6, 6, 6, 6, 6, 6, 7, 7, 10, 10, 6, 4, 6, 6, 11, 11, 9, 9, 6, 6, 6, 6, 5, 5, 8, 8, 6, 6, 8, 8, 6, 9, 10, 10, 6, 6, 6, ...


5

A) Here is the correct map: You made a mistake on your map at the PvuII site (it is not on 6.5kB from the start of the plasmid, but on 6kB). Can the Kpn I not go on the 8.5 site, it still creates the 2 and 8.5, so isn't there more than 1 correct option for plasmid map? Yes. What you need to do in order to make the correct map is try all possible ...


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 ...


5

Not sure why Larry Parnell was down-voted, he was not technically wrong. Crystal structures of the most popular restriction enzymes are already known and can easily be found the Protein Data Bank or Wikipedia for graphical reference. Any stretch of double-stranded DNA makes a complete 360 rotation (about it's helical axis) in 10-10.5 base pairs. A 180 ...


5

Such universal restriction enzymes would be very dangerous to leave lying around even if contained within a vacuole. Remember that bacteria are prokaryotes and have no nuclear membrane to protect their DNA. The analogy would be having a burglar alarm capable of burning down the house in order to protect it against thieves.


3

Note that a given bacterium will probably have more than one restriction enzyme, so the viral genome probably won't ever run out of targets. Even if it did, restriction enzymes could suffer mutations that may change the specificity. When you talk about microorganisms, it's more accurate to think in populations rather than individuals, since the colony would ...


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

Notice that viral DNA is not the only foreign DNA that a bacterial can meet during its bacterial adventures. Plasmids, conjugated DNA and DNA inserted by transformation exist, and it may confer ecological advantages. Because of that, even if the enzymes would recognize only non genomic DNA and destroy it effectively, it's still more useful to have some ...


3

Could be insert polymerization. If you have your stretch of DNA like this: (5')-AATTagctagcatcgtgatcgacg-(3') |||||||||||||||||||| (3')-tcgatcgtagcactagcagcGGCC-(5') And you take that, flip it around, it will ligate onto itself, like this: (5')-AATTagctagcatcgtgatcgacg-(3')(5')-CCGGcgacgatcacgatgctagct-(3') ...


2

I guess you are talking about the restriction modification system, not restriction enzymes in general (which are used a lot in the lab, for example). If so, this paper might help answer your question. I have not read through it totally, but at least some viruses trigger the downregulation of restriction enzymes to make them cleaving their DNA less likely. ...


2

I am not sure of a case where Taq doesn't add 3' A overhangs. You could use TA cloning to clone your PCR product. The basic principle is to use a vector with 3' T overhangs. If you have a vector without these overhangs, you can use a terminal transferase + dTTPs to create them (or even Taq, but this is not as great because it adds a purification step to ...


2

Restriction enzymes are only half of the system. The other half is the methylation system. The majority of restriction enzymes won't cut at their target site if it is methylated in the correct way. The system works to identify foreign DNA because the foreign DNA is not methylated in the correct way so it will be cut but the bacteria's own DNA is methylated ...


1

First, not all restriction enzymes cut at palindromic sequences. A lot of them do though, simply because it is more effective. Recognising a palindromic sequence enables them to cut both strands of DNA at the "same" site, because the strand will have the same sequence only in different directions at that site. See Wikipedia for example.


1

There are generic DNA cutting enzymes! E.g. DNase A (journal), DNase I (wikipedia), and NucA (Bacillus subtilis annotation). The outer layers of human skin contain active dnase, with just such a protective role as one apparent reason. This is one of the (many) sources of contamination that can mess up dna experiments.


1

@A. Kennard beat me to it. Here is my answer, assuming complete digestion. The two HindIII fragments can ligate to give four different products as shown below. I have marked one end of each HindIII fragment to help in seeing polarity, and I have left the ligated junction as a gap to help see what is going on. I don't understand the point about the 1 kb band. ...


1

You are being too smart for this question ;) If you assume that the enzyme is perfect, you've optimized the amount of template, and you leave the reaction for a really long time then there will be complete digestion. If you assume complete digestion, then there won't be any 1.1kb fragments. Furthermore, if you work out all the possible orientations of the ...


1

The answer from @Armatus got me looking at star activities again. EcoRI is prone to exhibit star activity in non-optimal buffers. In the case of this enzyme this tends to be a relaxation of site specificity, so HAATTC and GAATTD would be possible cut sites. The cleavage at such sites produces the standard sticky end. So, what if there is a star site near to ...


1

Use pGemT to clone your PCR product into. It is exactly designed to take products with 3' overhangs and features blue/white selection so is pretty easy to do! Then you would cut out of pGem with the restriction sites you added during your PCR and ligate it into your expression vector.


1

Most restriction enzymes recognize palindromes, hence the recognition site of an even number of residues. Why palindromes? This allows for a vast increase in complexity (or rarity) from a DNA sequence standpoint, while requiring very little added complexity from the protein. In other words, if a protein domain recognizes 2 or 3 or 4 bp of DNA, simply adding ...



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