I isolated my cloned plasmid from TOP10 cells using invitrogen plasmid miniprep kit and eluted it in TE buffer. But I noticed that contrary to my plasmid aliquot, it does not freeze at -20 degree celsius. I am asking this question because latter when i did confirmatory restriction, i am getting incomplete digestion, and i am linking it with the fact that my cloned plasmid aliquot does not freeze (i am questioning what else is there in it, or is it that i preheated the TE buffer at 70 degree celsius before elution step, so that affected the buffer in someway), thanks in advance for the guidance !!
It might just be your miniprep supercooling (when a liquid goes below its freezing point without turning into a solid because there's no nucleation point to initiate crystallization). It's common with small sample volumes because they cool down so quickly. Try flicking it a couple of times and see if it freezes suddenly.
What do your 260/230 ratios look like on your miniprep?
I think that you did not dry enough the coloumn after the washing steps and some traces of ethanol or isopropanol (those are commonilly prenset in the washing buffers) remains into your sample.
best
Manuele
Alexandra Johnson thank for adding to my knowledge about the supercooling aspect. I have tried that as well but ever since i isolated it, it stays liquid even if i check it after a day or two (last time i checked it 2 days ago)
and my A260/A230 ratio was 2.4
Manuele Martinelli you are right, that can be a reason for my aliquot not being frozen but my 260/230 were not low enough to indicate ethanol contamination. My value was 2.4 (which is still slighly above the recommended upper value)
Zaki Haider
As far as ethanol: I would expect a poor yield with significant ethanol contamination which usually correlates with a low 260/230 ratio, simply because there's not too much absorbance from the DNA at 260 nm and more DNA tends to be retained on the membrane in the presence of ethanol.
However, ethanol itself does not absorb very strongly 230 nm, especially in the presence of DNA, so a good ratio does not always exclude ethanol contamination, particularly if the plasmid is present at a high copy number. Absorbance at 230 nm is much better at detecting chaotropic agents meant to remove protein from nucleic acid preparations like guanidine hydrochloride and phenol. We've probably excluded chaotropes from being the culprit, at least. Do you have access to a vacuum microcentrifuge (sometimes called a SpeedVac)? If there is ethanol contamination you could remove it simply by evaporating the plasmid to dryness in a vacuum microcentrifuge then add a volume of purified water that is equal to the original pre-dried volume (since the Tris and EDTA will precipitate out also so you don't need to add more buffer) to re-dissolve the DNA.
Also, do you have another plasmid prep you can check this restriction enzyme with just to make sure it's not the enzyme itself going bad? What enzyme are you using?
Alexandra Johnson Unfortunately, i do not have access to the vacuum centrifuge, so i only have the spin column option for doing plasmid miniprep.
I doubt the enzyme having the problem as i have tried 2 different enzymes (which are available in my lab and with which i can confirm my cloned plasmid) and also i made another plasmid miniprep (eluting in prewarmed PCR water this time)
still i am getting partial digestion (at least this is what i understand after looking at the bands)
i tried doing confirmatory restriction with BamH1 and HindIII
If you have an image of the gel can you show us and let us know what the expected results are?
Alexandra Johnson yes sure ! attaching the image.
From left to right:
1. uncut plasmid
2. 1kb ladder
3. clone 1
4. clone 2
5. restricted plasmid
HindIII has 2 sites in my cloned plasmid (one in insert, one in vector) and my uncloned plasmid has only a single site. Now the uncloned plasmid is linearized but the cloned are not being fully restricted (that is what i think)