I’m trying to use alkaline comet assay on isolated rat lymphocytes and hepatocytes. But I couldn't get any usable results until now.
On the assay, I use high gelling temperature agarose (%0,75) for covering the slides and prepare the slides one day before use (I leave them for air dry at room temperature overnight). When I mix my cells with low gelling temperature agarose (%0,5) and put them on slides, I dry them at 4˚C up to 5-7 minutes to avoid humidity. Taking off coverslips without any problem and leave the slides in the lysis (2,5M NaCl, 100mM EDTA, 10mM Trizma base, %10 DMSO, %1 Triton-X) at 4˚C for 15 hours. After taking the slides off the lysis, place them in the electrophoresis tank, unwinding with alkaline buffer (10M NaOH/200mM EDTA) for 20 min and electrophoresis for 30 min. Then put the slides in neutralization buffer (0,4M Trisma base) and agarose gels drop off. Sometimes gel drop in lysis too but I think that is gel humidity problem.
I don’t understand where the problem is. What am I doing wrong? If you have any suggestions, please help me.
Dear Burcu Öztaşcı,
Slides in Lysis buffer for 15 hours ???...Seems too long...I think you will get good results if you reduce the time....Slides in lysis buffer for 2 hours in 4˚C should give you good results in case of lymphocytes.
All the Best !!!!
Hello, if this is the protocol you are using in your laboratory and someone else found good results using it then the problem is probably due to a wrong manipulation from your part. If not, then I think you should increase the duration of drying phase to avoid more humidity. Good luck.
Yes definitely allow the gels to dry. I used to have this problem and I started allowing the gels to dry for 20-30 min at room temperature before and after electrophoresis and this enabled better adhesion to the slides.
Dear Burcu Öztaşcı,
Slides in Lysis buffer for 15 hours ???...Seems too long...I think you will get good results if you reduce the time....Slides in lysis buffer for 2 hours in 4˚C should give you good results in case of lymphocytes.
All the Best !!!!
Dear Burcu Öztaşc,
Are you really use alkaline solution with concentration 10M NaOH?
Where you find such protocol?
300 mM NaOH must be used in all case!!
Hello everybody,
First of all, thanks to everybody for these good suggestions and second I wrote it wrong, I use 300 mM NaOH, my stock solution is 10 M, sorry... Actually someone get good results with this protocol but she can't help me now. So i will go over my steps. Maybe i should use oven on drying phase, and I will definetily try drying slides before and after electrophoresis. About lysis time, there is nothing I can do because lab is closed 17:30 and I'm not allowed to study after that. So this is the last thing that I'm going to try...
Try to work without neutralization. After electrophoresis put slides into alkahol. Dry them overnight and store it untill staining and scoring/
Dear Burcu Öztaşcı,
I think your exposure to lysis buffer is too long.
your process of solidification of gel may create the problem. try to solidify the gel in dry and cool condition. secondly duration of lysis is too long please reduce it.
Hi Burcu
You can try using a 1.5 % high gelling temperature agarose instead of 0.75%. for layering of cells please use 0.75% of low gelling temperature agarose. For protocol please refer this link ---http://www.cometassayindia.org/
I believe this modification will help you. I think a lower % of high gelling temperature agarose what you are using is not appropriate as a result of which you gel slips off. the other compositions of your buffers looks fair enough. please make sure that all the three layers of agarose what you make has solidified nicely. Once you make the first layer that with 1.5% high gelling temperature agaose, you need to make sure it has dried completely by putting the slide on a slide warmer which should be kept at 50degree centigrade.
An overnight lysis is good as it ensures that the lysis is done properly.
Goodluck
Regards,
Shubhankar
The exposure time to lysis buffer seems to be too long.the slides with initial coating should be dried at about 46 degrees and stored in a sealed box and used after 4 or 5 dayd
Hi Burcu,
I think these trouble shooting guide may help you. Thanks for your important and very relevant question.
http://www.scorecomets.com/about-the-comet-assay/troubleshooting-guide
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