In my cloning project I am digesting a plasmid then keeping the digested fragments in -20C after gel extraction. As the ligation of these double digested fragments (not blunt end fragments) have become a disaster for me, I'm curious if this freezing step can damage the sticky ends?
Hi Amir,
Usually, hydrolysis (and thus degradation) of DNA occurs after several freeze/thaw cycles. So I am not sure that freezing/thawing only a few times your plasmid will break the sticky ends.
In my own cloning cases, when cloning was a "disaster" as you said, it was due to another step which was not done properly, and not hydrolysis of DNA. So you should double-check your cloning procedures (is the double-digested vector gel-purified and dephosphorylated ? Do you manipulate properly the diffent enzymes ?...)
Hi Amir,
Usually, hydrolysis (and thus degradation) of DNA occurs after several freeze/thaw cycles. So I am not sure that freezing/thawing only a few times your plasmid will break the sticky ends.
In my own cloning cases, when cloning was a "disaster" as you said, it was due to another step which was not done properly, and not hydrolysis of DNA. So you should double-check your cloning procedures (is the double-digested vector gel-purified and dephosphorylated ? Do you manipulate properly the diffent enzymes ?...)
Hi Amir,
Pierre is right about double checking your cloning procedures. You also mention gel extraction. Are you measuring the amount of DNA you get out of the extraction procedure? Sometimes when cloning doesn't work and the concentration of the DNA hasn't been confirmed you're actually putting in less than you think into the ligation reaction.
Hi Amir,
In my experience, I routinely store my digested vectors and inserts @ -20 for extended periods with no problems. It sounds like yours may be an issue of troubleshooting the ligation. As Kiev has mentioned, it is important to know the concentration of your inserts/vectors so you can set up the adequate molar concentrations for your ligations. Also, make sure your ligation buffer is relatively fresh. If the ATP in your buffer is bad, your ligations are doomed from the start.
I would start with the simplest thing first, such as making or getting your hands on some fresh ligation buffer, and then work up to other manipulations of your protocol to very the molar ratios of insert/vector, and even play with the ligation times and temperatures.
Good Luck
Barry
I usually store DNA solutions at -20, and I did more than three freeze-thawing cycles. I did not come across any problem so far. I strongly recommend you to revise your digestion protocol and your purification step after digestion. Did you run your sample on agarose gel after gel extraction? Did you quantify your digested product after purification by gel extraction?
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