I agree, no way for a His tag (or any stretch of sequence) to disappear specifically and completely, from a plasmid upon storage. Most likely, the His tag wasn't there to begin with. Your sequencing results should give you an idea what has happened. Anything else missing? Somebody picked a wrong clone...?

Three ways for a correct plasmid to get unspecifically degraded upon storage:

1. Acidic hydrolysis (an often overlooked issue). Happens with time when the DNA concentration is very high, in a weakly or un-buffered solution, and/or when the DNA pellet was not washed sufficiently after EtOH precipitation. Acidic hydrolysis results in strand breaks (nicking), and subsequent open-circle conformation which will transfect poorly, esp. with large plasmids.

To prevent: After EtOH precipitation, wash the pellet and the side of the tube with a good volume of cold 70% EtOH, to get rid of residual Na-acetate. Never store your DNA in (unbuffered) water for a longer time. You want to avoid EDTA (as in TE), store in 10 mM Tris-HCl pH 8.5 without EDTA. Do not store plasmids at concentrations higher than 2-3 µg/ml. If unsure, check the pH of your final solution. It should be neutral or slightly alkaline, not acidic. If necessary, repeat EtOH precipitation.

2. Enzymatic degradation. Depends on the purity of your plasmid. In plasmids prepared using common "maxi" kits, this should no longer be an issue. If unsure, check uncut and single-cut plasmid on a gel. No smears, or multiple smaller bands should appear. In the uncut sample, a faint larger band might appear, representing nicked (open-circle) plasmid, moving more slowly in the gel than the supercoiled conformation. A small percentage of nicked plasmid is normal, and will not affect performance.

To prevent enzymatic degradation: Make sure your kit buffers (esp. elution buffer) are not contaminated. If you are using self-made buffers, use the purest chemicals, and the purest (commercial) water. Filter-sterilize or, if possible, autoclave your buffers after preparation. When purifying plasmids from large cultures, make sure your bacterial pellet is resuspended completely before you add lysis buffer. Increase the volume of lysis/neutralization buffers, to make sure lysis is complete. Also, increase the volume of column wash buffer. Make sure your final tubes are sterile and DNAse-free (autoclaved).

3. Storage conditions: Even at -20, a concentrated DNA solution might not be frozen completely, or it might go through freeze-thaw cycles. In this case, typical ice crystals will form at the side of the tube and near the lid, as a sign of "water turnover" in the tube upon prolonged storage.

To prevent: Make sure your freezer does not go through "anti-freeze" cycles, as many common household freezers do. Check the real temperature with a thermometer. If unsure, store your plasmids at -80.

Good luck!

In my expirience plasmids can be stored a -20 for years, we recently recover a plsmid that was constructed 15 years ago stored at -20 and was perfect. The problem you can have is degradation but not desapiering perts of it. Most probably it was already wrong when stored.

I agree, no way for a His tag (or any stretch of sequence) to disappear specifically and completely, from a plasmid upon storage. Most likely, the His tag wasn't there to begin with. Your sequencing results should give you an idea what has happened. Anything else missing? Somebody picked a wrong clone...?

Three ways for a correct plasmid to get unspecifically degraded upon storage:

1. Acidic hydrolysis (an often overlooked issue). Happens with time when the DNA concentration is very high, in a weakly or un-buffered solution, and/or when the DNA pellet was not washed sufficiently after EtOH precipitation. Acidic hydrolysis results in strand breaks (nicking), and subsequent open-circle conformation which will transfect poorly, esp. with large plasmids.

To prevent: After EtOH precipitation, wash the pellet and the side of the tube with a good volume of cold 70% EtOH, to get rid of residual Na-acetate. Never store your DNA in (unbuffered) water for a longer time. You want to avoid EDTA (as in TE), store in 10 mM Tris-HCl pH 8.5 without EDTA. Do not store plasmids at concentrations higher than 2-3 µg/ml. If unsure, check the pH of your final solution. It should be neutral or slightly alkaline, not acidic. If necessary, repeat EtOH precipitation.

2. Enzymatic degradation. Depends on the purity of your plasmid. In plasmids prepared using common "maxi" kits, this should no longer be an issue. If unsure, check uncut and single-cut plasmid on a gel. No smears, or multiple smaller bands should appear. In the uncut sample, a faint larger band might appear, representing nicked (open-circle) plasmid, moving more slowly in the gel than the supercoiled conformation. A small percentage of nicked plasmid is normal, and will not affect performance.

To prevent enzymatic degradation: Make sure your kit buffers (esp. elution buffer) are not contaminated. If you are using self-made buffers, use the purest chemicals, and the purest (commercial) water. Filter-sterilize or, if possible, autoclave your buffers after preparation. When purifying plasmids from large cultures, make sure your bacterial pellet is resuspended completely before you add lysis buffer. Increase the volume of lysis/neutralization buffers, to make sure lysis is complete. Also, increase the volume of column wash buffer. Make sure your final tubes are sterile and DNAse-free (autoclaved).

3. Storage conditions: Even at -20, a concentrated DNA solution might not be frozen completely, or it might go through freeze-thaw cycles. In this case, typical ice crystals will form at the side of the tube and near the lid, as a sign of "water turnover" in the tube upon prolonged storage.

To prevent: Make sure your freezer does not go through "anti-freeze" cycles, as many common household freezers do. Check the real temperature with a thermometer. If unsure, store your plasmids at -80.

Good luck!

it really depends on the plasmid type and plasmid structure, better would be storing at -80C. Supercoiled are more stable to my knowledge....but....It depends on the bacterial cells you grow to "produce" it. I find that once you have the plasmid is always better to retransform the cells rather than using old aliquota which are decades old but do not despair, for traditional cloning better use as fresh as possibnle, to avoid adding more variables but even for gateways there seem to be some issues.....In my experience gateway plasmis last for at least 1 year especially for the LR reaction, the BP a little bit less but I think it depends also on the quality of the preps and PCR product......

I have bacteria with plasmids stored for 5 years at -20 ° C, using a good cryoprotectant such as DMSO or glycerol in the culture medium at a concentration of 10% v / v

In order to make notice for the storage limit of your plasmid, you need to develop fast Uv-vis kinetics, if the UV-vis spectra is untouched and no variations of time zero and your unfreezing time hence your plasmid is fine to work with it. Otherwise, as prof. Ladero said, you should have the "parts" of it but degradation can occur. Hope this helps.

Thanks a lot for your responses. I also noticed that a single cut of the Plasmid has the same size as the uncut Plasmid on Agarose gel. But, i do get colonies upon transformation.

I do not get your part John. The plasmid generally runs in three bands. The plasmid is big, if the insert is small it is almost impossible to distinguish the biggest plasmid band with the uncut, plus since it is not linear it is not much clear the gel display.

if you do not get any colony try to change the media because the gene may be toxic but it is in the worst of the situation. I worked with one toxic plasmid once but it is rare.......Are you sure about the media? What info do you have about the plasmid? Please give more info.

One can store pasmid at -20 without any problem. Sometimes it is better to spot these on nylon or nitrocellulose membranes and store them dry

You can read this article it may help you or any other how face the similar situation