smear for digestion - (Nov/10/2009 )
phage434 on Nov 11 2009, 09:56 PM said:
The plasmid that I extract is low-copy number. If use 2mL will too little or not?
I am setting up 20uL of digestion reaction with 2uL buffer E, 0.2uL BSA, 0.5uL HindIII, 0.5uL BamHI, 5.0uL plasmid (~300ng/uL), dH2O.
But after i got a smear for it,i reduce the enzyme volume to 0.2uL.
I didn't use RNAse...I just follow the protocol of extraction kit... hmm...i think i will try to add RNAse next time...thanks ya...
Beside, still got any problem with my digestion?
I would immediately retry the digestion at lower concentration. You are digesting 1.5 ug of DNA in 20 ul, which is almost 4 times the recommended concentration of 1 ug in 50 ul. It's difficult to pipet accurately 0.5 ul of enzyme, so it is easy to approach too high levels of glycerol in a low volume reaction. Enzyme inhibitors may be present in your DNA preparation, which can inhibit cutting. With a higher volume, you dilute those inhibitors substantially (4x dilution in your reaction setup, vs. 16x dilution in a 1 ug, 50 ul reaction).