I have purified and isolated 50 - 100 kb and 120 - 200 kb DNA fragments from metagenome. I work with them separately. I know that so large fragments tend to shear if centrifuged. But is there at least any limit for gravitational acceleration used in centrifugation that would give intact (not sheared) at least smaller fragments 50 - 100 kb? Lets say I would like to centrifuge it out of 96 % EtOH na 2M NaAc solution or from 70 % EtOH.
Thank you for your answers! :)
There is no need to centrifuge the high molecular weight DNA. The precipitated filaments would be visible as you add ethanol (do not shake - just swirl gently !!). To avoid breaking DNA, you can collect it with "shepherd's crook" (a simple device with an U-shaped end that you can prepare yourself by heating a glass capillary or pasteur pipet, or disposable inoculation needle). If there is too little DNA to be seen (or the fragments are not large enough to precipitate as filaments), you can centrifuge 5 minutes at 5000 xG . This would preserve 200 kb large fragments. The 50 kb fragments would not break under conventionally high centrifugation speeds (>12000 xG), used, e.g., for isolation of phage lambda DNA (~49 kbp) :)
Depending upon the concentration of the large size DNA (such as bacteriphage lambda DNA), the DNA can be either centrifuged successfully at 1000xg without any measurable breaks or can be collected on a pipetman tip through winding. Important thing is not to let DNA dry completely. It may take few hours to couple of days to dissolve (hydrate) the DNA into appropriate buffer (TE) at room temperature.
Hello!
Thank you for your answers, they helped me! I would like to respond to them:
Mr. Burzynski, such as you, I do also have troubles to find some information about shearing of HMW DNA caused by centrifugation. But some people from our and other teams have this experience of shearing of DNA. Well, I do use PFGE and I isolate fragments of DNA that are pure for other downstream application. However, after many optimalisations I still do have problems with uniform restriction of the HMW DNA (stored in PFGE gel) to correct size. Therefore, I decided to restriction fragment HMW DNA in aqueous phase. To do that I need to concentrate it so I would like to use centrifugation after precipitation.
Mr. Khobta, you gave me good advice about "shepherd's crook" and similar accesory I thought that I would transfer DNA simply with sterile toothpick. Crook or sth. similar would be better. Can you please send me your article or some other work where was such high acceleration gravitational force used for centrifugation and HMW was not sheared? Thanks in advance! :)
Mr. Choubey, thank you for your information about dissolving the DNA. I also wanted to use TE buffer. I know DNA cannot fully dry but from the protocols that are on the internet I thought it would dissolve in TE in 24 hours. So I will see...
Thank you all for your answers, I wish you to have a nice Christmas time ;)
Thank you Mr. Spencer for good information to this problem! I have already been looking through some BAC and YAC protocols, it is very good source.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction