19

The 5' and 3' mean "five prime" and "three prime", which indicate the carbon numbers in the DNA's sugar backbone. The 5' carbon has a phosphate group attached to it and the 3' carbon a hydroxyl (-OH) group. This asymmetry gives a DNA strand a "direction". For example, DNA polymerase works in a 5' -> 3' direction, that is, it adds nucleotides to the 3' end of ...


5

I stumbled across this paper demonstrating it is between 150-200 base pairs. DNA flexibility studied by covalent closure of short fragments into circles D Shore, J Langowski, and R L Baldwin PNAS 1981 78 (8) 4833-4837 http://www.pnas.org/content/78/8/4833.full.pdf


4

The no 5 and 3 are the carbon no of the carbon skeleton ring of deoxyribose as similar as any other organic compound. In any nucleic acid, RNA or DNA 3' refers to the 3rd carbon of sugar ribose or deoxyribose which is linked to OH group and 5' linked to a triple phosphate group. So these 5' and 3' group provide a directional polarity to the DNA or RNA ...


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') |||||||||||||||||||| ...


3

The best resource for troubleshooting ligations I found (and use frequently) is this NEB page. That is assuming you've already referred to the instructions provided with the enzyme you're using. In my case it's T4 Ligase, again from NEB. It's helpful to check the FAQs and the references listed on that page. Also, you can make use of their Molar ...


2

For ligation of a linear molecule to occur, the two ends must come together at the active site of the DNA ligase. In a simple molecular cloning experiment the aim is usually to avoid recircularisation of a cut plasmid vector, and instead to get a new fragment of DNA inserted into the vector. However because the ends of the linearised circular plasmid are ...


2

As per WYSIWYG's suggestion, I tried the digest and dephosphorylation again with SalI and ran a gel. The gel showed that SalI was less than ideal at cutting this plasmid. As you can see, the most intense band is the bottom band, which is most likely supercoiled DNA, with the linear DNA I want appearing in the middle, and open circle DNA coming in third. I ...


2

From my experience I would say that it is no problem to freeze the digested DNA, when the enzyme is inactivated (even when not, for this time period I wouldn't expect much damage). However, if possible, I would rather opt to freeze the PCR product and make the digest fresh, since the overhanging ends are kind of sensitive to hydrolytic degradation. I have ...


1

So, after the restriction digestion, the sample consists of DNA fragments with 5' overhangs. These are filled in by Klenow DNA polymerase, adding biotin-dCTP to both ends of each fragment. Then the fragments are ligated under conditions (ie low DNA concentration) such that ligation between non-crosslinked DNA is less likely. This reduced ligation efficiency (...


1

To measure the frequency of indels at the ligation site you can use a vector with a unique restriction site in the lacZ gene. With a colorimetric assay you can count the number of white cfu. Perfect ligation in-frame yields blue cfu


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