non-specific bands in agarose gel - too many bands visible! (Aug/29/2005 )

hi guyz,,

I am working on pJOE 4036/pET 21a and TvDAAO/pET21a plasmidsfor two weeks.

i did a Miniprep( qiagen , digested it with NDE I and BAM HI restriction enzymes for 2 hrs, used fermentas buffer tango , inactivate the DNA's at 65 celcius for 20 mins , and transferred it on to a 1% agarose gel .

Since i used only two REnzymes i am only supposed to get two lines, instead i see 4 lines.For pJOE (DNA) should lie around 4.5k bp and insert around 1.1k bp.

could it be possible there is contamination ?

please share some tips.

What was the concentration of each enzyme and total volume of the reaction? Have you checked the conditions of the reaction properly? Both NdeI and BamHI can exhibit Star activity under certain conditions.

Have you tried doing single digests of each enzyme to see if each is cutting the correct number of times (the vector would be linearised ie: 1 band visible)

The most likely causes of your problem are:

1. Incomplete digestion
2. Star activity
3. Incorrect sequence

I would expect that 1. is the most probable, especially is the extra bands are larger than 4.5kB.

Daniel

Molecular biology advice

the concentration of my enzymes 20 U/ µL which is 2 µL in a total reaction volume of 50µL. volume of dNA is 20 µL .

if i were to lengthen my digestion time wouldnt that increase more bands.i can show you my results .

I will try now with single enzyme digestion.see if it works.
Thanks a lot though for your help.