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7

The Qiagen manual for the Miniprep Kit states that buffer N3 contains guanidine hydrochloride and acetic acid. The MSDS for buffer N3 gives a bit more information: Guanidinium hydrochloride 25-50% Acetic acid 10-25% pH 4.3 Buffer P3 uses 3M potassium acetate, the potassium is usually important in plasmid preparations as potassium SDS is used to ...


4

This is particularly an issue when you do maxipreps and the pellet is 200x the size of the pellet from a miniprep. The reasons why? Maybe you're pelleting at too high of a g-force (pure speculation). Alternatively, you could be growing the cells too long. I typically miniprep after a 12 hr culture. If you're seeing any darker colors in your pellet, you have ...


4

What I would usually do is transform E. coli with the plasmid, grow an overnight culture and mini prep the plasmid in the morning. Before you do the miniprep save a glycerol stock. Thus, you would have some plasmid to work with in the next couple of weeks, and also a glycerol stock to come back to. You can also keep the plate with your transformants, as you ...


2

We routinely use E. coli preps from 16-18h overnight cultures. The cultures don't show any overt signs of lysis (sometimes you can see cellular debris if the culture has indeed overgrown). I have also noticed inconsistent times when resuspending the pellet. For my own use, centrifuging at too great a g-force or for too long can increase the likelihood of ...


2

What I do to avoid this is to ressuspend the cells by pipetting up and down the P1 (ressuspension) buffer on the side of the tube. This way, every time takes only a bit of cells every time and big clumps are avoided. The only times I used to get big clumps hard to ressuspend were when I went poking at the pellets first.


2

N3 and P3 are different. N3 contain chaotropic agents. P3 does not. Mainly there are two types of DNA purification kits on the market: silica membrane (glass fiber) based and DEAE anion exchange based. The silica membrane based method requires high concentration of chaotropic agents (salts that can change the water structure dramatically, GuHcl, GuSCN, NaI, ...


2

Seems like you have covered the essentials here. I can't think of anything else. Since the Qiagen patent on spin prep columns ran out, these kits are very cheap - $0.40 each? In the 3 or 4 kits Ive used, they all seem to use the same protocol and about the same buffers, so there might be differences in quality or yield but if so, they are small. You ...



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