double digestion with BamHI and HindIII - (Jan/04/2007 )
Hi,
I think that your problem is your DNA concentration and the time's restriction. Maybe you put too much DNA and restrict no enough time. Try to quantifie your DNA on agarose gel or by spectro!
With new england biolabs you can restrict your DNA O/N without any problem.
biofred
well, assuming a relative bad preparation, you should have 400ng per µl
so 30µl are 12µg so it is too much.
BSA addad seems to be too much too, except if it is 10x concentrate
finaly you can digest overnight with hind III.
For bam H1 it is more touch.
You can use a pcr equipment to do a 4h digestion starting in the evening if you want to save time
I think that your problem is your DNA concentration and the time's restriction. Maybe you put too much DNA and restrict no enough time. Try to quantifie your DNA on agarose gel or by spectro!
With new england biolabs you can restrict your DNA O/N without any problem.
biofred
Pardon my ignorance, how can you measure DNA conc by gel. Wouldnt it give an approximate value......Spectro machine in lab also gives me two different values for same sample.........
Thanks
Use ladders which give u DNA conc. for each band ( 2log DNA- NEB, or lambda HInd3 ladder).
For eg. if u load 1 ug of 2 log DNA ladder in a lane, u should c a 1000b band - which should b around 120ng. Now u would compare the intensity of ur DNA sample running alongside the ladder and if the intensity is similar to the 1000bp band, then u have 120ng. Normally I would run 2 different conc. of the sample DNA to get an approximate conc. value. There r different sized bands and there would b different conc. of each fo them.