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1

If you don't want acrylamide in your preparation, don't use it, as you will always have some carry-over in the solution. And you will not get rid of it completely, as it at least partly co-precipitated with the nucleic acids (it's used as a co-precipitation agent for this purpose). I think the best solution is to use chromatography, either size-exclusion ...


3

Just to add to the above responses, you are better off not use MQ because if it is contaminated with DNases, then it will digest your construct if it reaches room temperature or 37 oC as you are melting the frozen construct in your hand! TE-buffer is the standard buffer for DNA elusions but make sure your column is compatible with it. For transfection based ...


5

The Quiagen kits I'm familiar with explicitly state that you can use water instead of elution buffer for elution. The choice of continuing to use buffer or water is yours and depends on what you want to do with the DNA and whether the buffer interferes with later steps. Using the buffer is probably safer for long-term storage, but I don't know how much this ...


6

I would generally recommend using the elution buffer (which is typically Tris-EDTA buffer) or TE-Buffer, as the pH and the conditions stabilize the DNA for a longer time, which is especially interesting when you want to store your DNA. It also dependent on your downstream application - when they recommend diluting the sample in water (or another buffer) I ...


0

I'm not quite sure what you are doing. Are you running the whole gel each time and cutting off the lanes you've used, or only putting the lanes you desire into the tank and leaving the rest outside? If the former then the ethidium bromide will be leaching out of the gel because it is positively charged so each time you apply a current to it some of it ...


3

This is too long for a comment, so I post my thoughts about this here: This falls into different important parts here: One is an insufficient understanding of the techniques on which the method is based. This makes it hard to decide which steps are important and which are not. One prominent example would be the use of EDTA in DNA gel running buffers. This ...


1

Why do a gel purification? you can just use a kit similar to QIAGEN gel extraction kit and do a non gel based plasmid purification and elution, using the buffers used as usual! PCR DNA clean up kits will do as well!


2

Can't exactly say what problem you are facing but you can follow these steps for efficient restriction digestion and elution. Never set up a digestion reaction with more than 1µg DNA per 20µl; if you require more digested DNA, then set up multiple 20µl reactions with maximum of 1µg DNA in each (500-700ng is ideal). 10µl enzyme for a 230µl reaction: bad ...


0

Yes, fixation of almost any kind can have effects on morphology. When you take a free flowing, protein spiked fat-blob (ie cell membrane), and make it rigid, you are going to get some differences. A fun visualization of this can be done by wrapping cellophane/shrink wrap around a serological pipette and dipping it in a dry ice and methanol bath. It's ...



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