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How long can I store cleaved/digested vector?


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#1 linli

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Posted 16 February 2011 - 02:28 AM

Hi,
Does anyone know how long I can store a vector that has been cleaved with restriction enzymes, analysed on gel and then purified and eluted in dH2O. The vector is stored at -20˚C. I know that linear DNA is not a stable as circular DNA. I would like to be able to use my restricted vector for ligation with a new PCR product, some weeks later then I did the restriction. Is that possible? How long can I store linear PCR product that has not been cleaved?

Thank you!
/Lina

#2 SOS response

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Posted 16 February 2011 - 05:18 AM

I would say months, it is stabile enough, you can do as you planned. pcr products can also be stored for a long time. In my lab, college store his plasmids on room temperature and he has no problems.

#3 phage434

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Posted 16 February 2011 - 05:38 AM

Make sure you are storing in TE rather than in something like RE buffer. What we do is to PCR amplify the vector with primers to the MCS region, including the left and right restriction sites (you can change them easily at this stage as well). We cut the pcr product with DpnI, which eliminates the uncut circular template, making the DNA non-transformable. We then purify and store the linear (but uncut) vector as a stock. For cloning, you are typically setting up RE digests. Simply set up one more with the linear DNA. When the digests are done, mix, ligate, transform and go. If you are cloning a single insert, the mixing can be done before the RE digest.

#4 linli

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Posted 16 February 2011 - 06:57 AM

Thanks for fast answers. I use to store my DNA in water, no buffer, does that decrease the time I can store my vector?

#5 phage434

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Posted 16 February 2011 - 06:47 PM

Yes, dramatically, for two reasons. Pure water absorbs CO2 from the air and becomes acidic, which trashes DNA. And small amounts of contamination from fingers, e.g. will add magnesium, which acts as a co-factor to many nucleases. TE buffers at pH 7.5, and has EDTA to chelate the magnesium.

#6 linli

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Posted 17 February 2011 - 04:56 AM

Ok, thanks for the information. I've only learned to use water and that buffers could cause problems in later cloning. The elution buffer that is included in DNA extraction kits from Qiagen or Fermentas are 10mM Tris-Cl pH 8.5. In the manual it stands that TE buffer is not reccomended because EDTA may inhibit subsequent enzymatic reactions. If you use TE buffer have you had any cloning problems? If I use elution buffer from the kit and later do ligation or digestion with high DNA concentration (need to add a lot of DNA because my DNA concentration are low)with means high elution buffer concentration. Can that be a problem for enzymes (restiction enzymes, T4 DNA ligase or polymerase) or during transformation of competent cells? Can the high pH 8.5 be a problem?

#7 phage434

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Posted 17 February 2011 - 08:11 AM

It is best to avoid making the majority of a restriction digest volume from DNA, because it often contains contaminants. Increase the volume of the digestion, if necessary, or concentrate your DNA further. The impact of TE on restriction digestions is minor, which you can see just from looking at the relevant concentrations. TE has 1 mM EDTA, which can chelate (even if present at 100% in the reaction) a maximum of 1 mM of magnesium. A typical restriction digest buffer has 10 mM or more magnesium, so the influence of the EDTA, especially when diluted 2x - 3x is very minor. Similarly for the effect of the 10 mM Tris buffering in the presence of much larger amounts of buffer at higher pH in the restriction buffers.

#8 linli

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Posted 18 February 2011 - 05:12 AM

Thanks again. I know that it is not good to have a too high DNA concentrations during retriction digestions, but sometimes it happens. When I checked how I do I discover that during restriction I have maximum 20% DNA in the sample with means adding 20% TE buffer to the sample which should be OK. However during ligation I have 50% DNA in the sample. Is it still OK for T4 DNA ligase to have 50% TE buffer?

#9 phage434

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Posted 18 February 2011 - 05:29 AM

Do the math: you now have 9.5 mM magnesium instead of 10 mM magnesium. This is probably not going to change anything.
You can also switch to 0.1 mM EDTA in your TE, which is sometimes used instead of 1 mM, to reduce the effect of large amounts of DNA in reactions.

#10 linli

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Posted 18 February 2011 - 06:08 AM

Ok, thank you! Have a nice weekend!




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