double digestion problem - (Mar/16/2005 )
i am doing double digestion-Bam H1 and HindIII(biolab), HindIII is NE buffer 2, Bam HI is unique NE buffer. even bam H1 is 100% activity in NE2 buffer, i did double digestion same time in NE2 buffer. seems digest not completed. the catalog of Biolab suggested Seq digestion. It will take two days and after each digestion, clean up will lose 20%. finally the band is so thin, even hard see on the gel. any suggestion?
drive me mad
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.
I did double digestion in NE buffer 2 overnight in 50ul.
after digestion, there are two bands of plasmid digestion.
wow... i assume that digestion is not 100% but overnight...
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...
I directly use the Bam H1 buffer supplied from biolab, is it BSA included?
i always add bsa when using this buffer... I don'n think bsa is pre added...
Hi Cathy. I would definitely do sequential digestion, but you do not need to clean up in between. look at the composition of both buffers:
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 voilà, 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-online.org/forums/inde...pic=4961&hl=bam
Hope that it helps! Cheers
thanks so much, badcell and fed 33. really helps!!!! i will do it today! by the way, the digestion at least how many hours? after first HindIII digestion, do i need to inactivate HindIII 65 20mins? and how much is the minimum digestion volume you can set and enough for gel extraction and later for ligation?
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.
Thanks million just learned this word here, haha. I am running the first digestion, second one tonight. fingers crossed!!