gel extraction problem

[biolog35]

hi all,
after partıla dıgestıon and gel electrophoresis of dıgestıon product ı performed gel extractıon to get desıred bands but ı lost most of DNA .so ı could not perform clonıng:( please help me.. ı use ınıtrogen gel extractıon kit.

[victor]

Maybe the time is not long enough when you incubate the washed DNA with eluent buffer. In my lab, most staffs here use QiaGen QiaQuick Column and mostly they succeed in DNA extraction from gel.

[canalon]

More details would be graet to trouble shoo t your reaction, but the main problems I know with kits are poor lysis (depends on your starting material, the condition in your kit are not always optimal) and poor elution. Usually because of elution with milliQ water, it is acidic, and elution is much more efficient at pH between 7 and 8. 10mM Tris pH 7 is way better.

[victor]

hmm..I should note that when I extracting DNA
Thanks.

[integreti]

Most of the times, you lose DNA at the binding step itself- If you use TAE gels, typically the pH is ~8.0. But the binding requires pH<= 7.5. Although you use 3-5 vol gel-dissolving buffer it does'nt help much most of the times. Adding 5-10 ul of 3M sodium acetate pH5.2 will aid in better binding of DNA to the column. I always add 10ul NaAcetate to the sample before application on the column, and use MilliQ water to elute DNA. Have'nt had less than 90% recovery so far.