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1

The basic steps of ChIP-Seq are: Crosslinking proteins to DNA - this fixes the proteins in their natural positions Nuclease digestion - this removes regions that are unbound to protein; nucleases are sterically hindered from digesting protein-bound DNA Immunoprecipitation - this allows isolation of the target protein by binding it to a selective antibody ...


2

So that's not quite how ChIP-Seq (Chromatin ImmunoPrecipitation and Sequencing) works. First, you start out with a large number of cells per sample (preferably on the order of 107 or more - the more sample, the more immunoprecipitations you can do to different targets) and do whatever it is that you're going to do to induce transcription factor binding - ...


2

I always add the Taq to the mastermix. First it makes the handling easier and it avoids pipetting steps which can cause contamination and can also be forgot. Then the enzyme is very stable and will even tolerate room temperature without problem. Since we are going to heat this 30-40 up to 95°C, so this is clearly not problematic. Since the reaction mix is ...


2

If it is a good idea to use PacBio depends a lot on what you want to sequence and what you want to do with the data you get. In short: you get longer reads but about 15% error per base. This means, you will have to cope with those errors, there are possibilities to do that. How much coverage you will get of you sample I cannot tell you, as I do not know what ...


6

You can run your DNA sample on agarose gel to see, whether you have significant degradation. If you are interested in contamination, you can make a standard photometric analysis to assess the 260/280 and 260/230 ratios and absorbance at 320 nm on NanoDrop or even something similar to Eppendorf's BioPhotometer. In case RNA may be an obstacle for some ...


5

Yes, you can do this. As long as the final mix has the proper concentrations of everything, its fine. Just make sure you compensate the by adding 30 uL (the extra volume) less of water to the master mix.


5

No, it isn't that simple, because centrifugal force varies with the square of the rotor speed. Have a look at the Wikipedia page for clearing factor. Those equations are usually used to determine what you need to do if you are going to use a different rotor, but they can also be used to solve your question. Because you are looking at a situation where the ...



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