double digestion problem
Posted 16 March 2005 - 06:32 AM
drive me mad
Posted 16 March 2005 - 06:52 AM
how long was your experiment ? i would try an overnight digestion in neb buffer 2 in a volume not inferior at 50µl
If it's not solving your problem, it seems you don't have the choice...
but alcool/salt precipitation is not a bad method in double digest and i don't loose more than 5% of dna after the frst digestion. Could be a possibility for you.
Posted 16 March 2005 - 06:57 AM
after digestion, there are two bands of plasmid digestion.
Posted 16 March 2005 - 07:06 AM
well all i can say is that if you want to save time, start digestion with bamh1 at 9 in morning pellet at about 2pm and you can start the second digestion overnight...
did you use BSA? Bamh1 need bsa for digestion...
Edited by fred_33, 16 March 2005 - 07:08 AM.
Posted 16 March 2005 - 07:57 AM
Posted 16 March 2005 - 08:32 AM
Edited by fred_33, 16 March 2005 - 08:32 AM.
Posted 16 March 2005 - 08:59 AM
NEB buffer 2: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2,1 mM DTT, pH 7.9
NEB BamHI buffer:150 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2,1 mM DTT, pH 7.9
What you need to do is to set up the Hind III digestion first, incubate for several hours, and then add enough NaCl to the digestion so as to increase the 50 mM NaCl in the buffer NEB2 to 150 mM final NaCL, and voilą, you've got NEB BamHI buffer! Then you only need to add BamHI and incubate for a further several hours. Done. Be sure to use an NaCL stock solution enough concentrated such as not to dilute much the other components in the digestion. For instance, if you digestion volume is 50 ul, you'd only need to add 1 ul of NaCl 5M, so it will not significantly affect the concentrations of the other reagents in the mix. I do this all the time when I do sequential digestion, I always made sure that I do first the digestion with the lower salt content in the buffer. It works fine, and you don't loose time dyalizing or precipitating the sample.
Also, I never bother in adding BSA, even for the RES which supposedly need it, but according to the NEB catalog, BSA would never hurt, so if you want to add it, you can add it already to the first digestion.
You can also check this previous thread: http://www.protocol-...pic=4961&hl=bam
Hope that it helps! Cheers
Posted 17 March 2005 - 02:12 AM
Edited by cathy, 17 March 2005 - 02:19 AM.
Posted 17 March 2005 - 04:06 AM
I use to leave my digestions for at least 5-6h. For convinience, when I do sequential digestion I incubate the first one during the day and the second one overnight. You don't need to inactivate HindIII after the first digestion; its activity will decrease during the second digestion when you add NaCl to accomodate BamHI; but you can inactivate it if you want. I usually set up my digestions in 40-50 ul final volume, using around 2 ug vector DNA (usually 3-5 kb in lenght). That's enough to get a lot of DNA after extraction.
Edited by badcell, 17 March 2005 - 04:06 AM.
Posted 17 March 2005 - 05:14 AM