Agarose Gel Analysis Of The Purification Procedure

Evaluate plasmid DNA quality by agarose gel analysis

DNA yields and quality can be readily analyzed by agarose gel electrophoresis. Poor yields and quality can be caused by a number of different factors. To determine at what stage of the procedure any problem occurred, save fractions from different steps of the purification procedure (see table Sample volumes required for agarose gel analysis), and analyze by agarose gel electrophoresis.

Preparation of samples

Remove aliquots from the cleared lysate (sample 1), flow-through (sample 2), combined Buffer QC wash fractions (sample 3), and Buffer QF/QN eluate (sample 4), as indicated in each protocol and in table below. Precipitate the nucleic acids with 1 volume of isopropanol, rinse the pellets with 70% ethanol, drain well, and resuspend in 10 µl TE buffer, pH 8.0.

Sample volumes required for agarose gel analysis

Sample Protocol step Midi Maxi Mega Giga (Very low-copy
plasmids/cosmids)
QIAGEN-tip 100		 (Very low-copy
plasmids/cosmids)
QIAGEN-tip 500
1   240 µl 120 µl 120 µl 75 µl 600 µl 750 µl
2   240 µl 120 µl 120 µl 75 µl 50 µl 24 µl
3   400 µl 240 µl 160 µl 120 µl 200 µl 120 µl
4   100 µl 60 µl 22 µl 20 µl 50 µl 30 µl
(% of prep represented
by each sample volume) 		2% 0.40% 0.08% 0.02% 1% 0.20%

Agarose gel analysis

Run 2 µl of each sample on a 1% agarose gel* for analysis of the fractions at each stage of the plasmid purification procedure. This figure shows an analytical gel of the different fractions, together with examples of problems that can arise at each step. If you find that you have a problem with a particular step of the protocol, turn to the hints in the relevant section of the troubleshooting guide in the handbooks. If the problem remains unresolved, or if you have any further questions, please call QIAGEN Technical Service.

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.