Of course, a DNA topoisomerase produces multiple bands corresponding to topoisomers, DNA having different linking number. But, to observe these multiple bands, it is necessary to perform the electrophoresis properly (quality of agarose and %, appropriate buffer, electric field…). If the electrophoresis is OK but you observe only one band, may be you have a contamination with a nuclease which leads to the formation of nicked DNA (open circular form or FII). Be careful, we have a lot of nuclease in our hands etc… same problem than RNAse…

Finally, concerning the buffer, which topoisomerase I you used ? If it is the eukaryotic enzyme, the final buffer usually used contains about 50 mM Tris-HCL with a pH 7.5-8, 50-100 mM KCl and the addition of Mg increases the activity but is not essential for the reaction.

thank you Marc , Could you please look through this gel. I ran gel electrophorosis and been facing this problem lately. I am not able to achieve 92% of supercoiled DNA. The plasmid DNA i used is brand new. I got it two days back and ran in agarose gel but I saw 30% relaxed form and 70% supercoiled dna. For my experiment , I need more than 90% supercoiled dna. At first i thought , it might be due to the sterile water , that i used for dilution and didnt add (but didnt get result) later i didnt add loading dye but no changes . In fact i got two vials of PhiX 174 RF I DNA and both are showing same percentage of relaxed 30% and supercoiled 70%. I am attaching the doc file , so that you understand better . Thanks again. 

PS I used gloves while doing all experiments so i dont think there is nuclease contamination 

HI, Michael, I have bought these Plasmid PhiX 174 RF I DNA from NEB( New England Biolab) I have not done any cloning and purification of plasmid. 

Thank you 

Hi Thupten,

I have a look on your gel. The electrophoresis is not very good and your interpretation is probably wrong. You are right for the n° 3, it is the supercoiled form but the n° 2 is probably a mix between the open circular form and the supercoiled dimer. Consequently, the n° 1 is probably the open circular form of the dimer. There is no topoisomer on this gel but you don't incubated with a topoisomerase, it's right ? You will observed topoisomer after incubation with a topoisomerase or if you want to see the topoisomers contain in the supercoiled form, it is necessary to use gel containing chloroquine.

For the electophoresis, reads my paper, for exemple Bizard et al , J Mol Biol, 2011.

To disolve agarose (1.2%), never used a microwave… prepared the agarose gel each time… and used a TEP buffer (the best with my composition, not the method described in the Maniatis) or TAE (requires a longer run and not the best), do not use TBE buffer. It is important to have a thick gel and to migrate slowly.

Don't hesitate to contact me after you read the M&M of my papers.

Thank you , Marc Nadal. I read your paper."Insight into the cellular involvement of the two reverse gyrases from the hyperthermophilic archaeon Sulfolobus solfataricus"

No , I did not use Topo I in that picture, I wanted to show you that , I get three bands . ( Which means that I have either nicked/ relaxed form) also, the supercoiled form is showing less than 75% . I will try with thick gel today and run in TAE buffer. What do you use to dissolve agarose ?

Hi Thupten,

I usually perform electrophoresis in a 1.2% agarose gel in TEP buffer (36 mM Tris, 30 mM NaH2PO4, 1 mM EDTA, pH 7.8) and the gel runs at 3 V/cm for 6 h (3V/cm max, it is possible to run at 0.9V/cm overnight or it is also possible to run for only 3 hours at 3V/cm for a rapid screening of the conditions). Before staining, the gel is washed in TEP buffer for 30 min and stained with ethidium bromide (2 µg/mL for 30 min) and digitalized. If you used TAE instead of TEP never run at a higher electric field than 2-3 V/cm and the run take more time. The agarose is dissolved in the buffer (TEP or TAE) on a heating magnetic stirrer until boiling for 2 min then cooled at temperature about 50-55°C (until you can take the erlen in your hand).

Hi Marc Nadal,

I use 0.8% agarose and run In TAE buffer ph 8. I think stock DNA (PhiX 174 RF I DNA) is already in damaged (coz it shows 25% relaxed form and less than 75% supercoiled form). Therefore, I put request to NEB to exchange or give new vials of DNA. Hope they accept my request. Thank you so much , I really appreciate it.

Thupten 

I use the same DNA but the supercoiled form is more than 90-95%.

oh  from which company you got your PhiX 174 RF I DNA ? New England Biolab? and which software do  you use? Lab image ?

what could be the cause of getting less supercoiled form? i know that, repeating no. of freezing and thawing could damage the dna. But after getting new DNA , I immediately ran agarose gel still, supercoiled form % is less than 75%.  In addition, while loading , pipetting, etc. could damage dna. Any chance that ultra pure milliQ ph 6.3 might effect the conformation of dna/ damage the dna? Loading dye might impact on conformation?