Is it possible for me to freeze my product after performing a restriction digest so that I can continue working tomorrow? Or is it better to place the product at 4°C?
4c will also be fine. the products are stable aand sterile. freeze thawing will sometimes break up dna randomly so I prefer just cold to frozen but it will not make much difference bot if you freeze it then mix gently after thawing or you may get local concentration differences as thawing is an uneven process
If you don't have time to gel purify your reaction you can still put it in the freezer overnight. If the enzyme is still active I wouldn't put it at 4C. You might want to heat-inactivate the enzyme to avoid star activity while you are freezing/thawing - generally by heating it to 65/70C for about 20 minutes. Some enzymes can't be heat-inactivated, so check the data sheet for each enzyme.
When I have to store my digestion for the next day firstable I make a heat inactivation (it is specified in your enzyme datasheet) or an EDTA inactivation (if your sample can't be heat inactivate). Then you can store it a 4°C, often freezing can break DNA in some point.
I know this is off-topic, but I have to say I am surprised to be told that freezing will break DNA. This isn't something that I've ever observed - at least, I've never seen fragments in a gel, for instance.
After restriction digestion, run the gel and elute your known band then eluted product again check it in gel and you can store it in -20 C. If you store it in -20 after restriction digestion, if any contamination or salts may degrade the DNA
The product of restriction digestion can be easily stored at -20 C. At 4 C it would be fine but to ensure that there is no activity and no star activity it is recommended to keep it at -20 C. Be careful, for the rest of your experiment, based on what you are going to do, you need to deactivate the restriction enzymes by for example heating at 60 C for 15 min. For other purposes you d better to purify your product.
I would freeze (to avoid any star activity - see above - that might (or not) be present in the particular RE you have used) but after thawing, vortex mix very well as the solute (DNA) will be concentrated at the bottom of the tube as the water in the solvent freezes out.
Given these exchanges, can one of those who thinks that freezing and thawing DNA can fragment it give me a reference to the study that shows this to be the case for DNA less than 10kb.
How long can a double digested plasmid be stored after cleanup/purification? Is it okay if i store the double digested plasmid in -20deg after cleanup and use it after few weeks?
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