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smear for digestion - (Nov/10/2009 )

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phage434 on Nov 11 2009, 09:56 PM said:

That's some information, but not enough. I would replace the water for elution with TE. 10 ml of culture sounds as if you may be overloading the column -- try 2 ml instead. What volume are you doing the restriction digest in? Too much DNA in low volumes can lead to problems. Aim for 100 ng/ul DNA concentration from your prep, and cut 10 ul (1 ug) in at least 50 ul of restriction digest. I'm guessing the problem is overload of your column and extraction of genomic DNA during your "plasmid" prep. Also check to make sure that you have added RNAse to the initial resuspension buffer -- the smear could be RNA coming through the miniprep. What enzyme are you using? Buffer? Amounts? Do you add BSA? We need to know it all.

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).

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