Hi Guilherme,

You should use TBE, because, the conductivity of the buffer is important for electrophoresis. If you use mili-Q water which is a very low level of conductivity, your electrons are not going to move through the agarose gel properly. This could probably effect your migration results. 

If you want to clear slides after drying. First of all, prepare your NMA agarose in a microwave. Then, You should keep your normal melting agarose in a slide jar inside a water bath which should be set 42-45 C for 30 mints. Put a thermometer inside the jar, make sure your agarose at 42-45 C.  When you cover your slides with this warm NMA, Just turn them upside down and put them vertically until they become solidified 30 minutes at RT. (You can use a slide holder to keep the slides vertically) After that keep them at +4C.   

I hope this helps

Good luck 

Dear Cigdem, thank you for your answer!

We have made some slides today with milli Q water, I will tell you how it went after we stain it. We thought that maybe using only the LMA with TBE would be enough for the DNA to migrate.

If you leave the slides to dry in vertical position, doesn't it make the agarose to be uneven distributed, thicker in the bottom part of the slide?

Also, are you using two or three layers of agarose in your slides:

1 - NMA, 2 - LMA + cells, 3 - LMA; or

1 - NMA and 2 - LMA + cells?

Dear Guilherme,

you are right about migration inside LMA, after I wrote the answer I thought the same.

If you do the manoeuvre very quick, it is not get thicker at the bottom of the slide.

I use 3 layers, it works perfectly.

good luck

Dear Mr. Wood, 

I've been using alkaline comet assay for 7 months , the whole process is very simple . all what you need to coat your slides with the first layer is 1 gram of NMPA and 100ml of normal distilled water . mix them in a beaker and dissolve untill boiling in a microwave oven then roughly  stir  the hot solution until it gets completely clear. put it in the 60°C water bath for 10 min then dip your clean frosted end slides in the solution with some sterring. wipe the underside using a toilet paper and lay the slide on a toilet paper spread on an even surface  to dry. Leave your slides like this overnight then store them at room temperature ! !. yeah , at room temperature . (When they are dry, they will look clear as if they were not coated at all. but it is OK, there is agarose ) . 

In addition , you definitely ain't in need for a third layer of agarose.

Best wishes , and let me know if you hit the point . 

Thank you for your answer Mohammad!

After running some tests, we noticed that you are right - it looks like diluting NMA with TBE or water does not affect the final slide. We also noticed that slides with TBE are cloudy before electrophoresis, but they get clear in the end, so it won't affect microscopy. Also, the third layer of agarose doesn't seem to affect the final product.

Now we are facing another problem: we are using SYBR green to stain our slides, then we wash it with TBE and cover with a coverslip. We noticed that the slides are good in the first day, but after that they are unreadable: we see everything green in the microscope. Has any of you ever had this problem? Maybe it is the SYBR green staining our coverslip? Agarose that is outside the coverslip is readable.

Dear Wood, 

First, you don't need to wash your slides with TBE after staining. chilled distilled water is sufficient for this purpose . and try to wash well enough to remove excess stain.

Second , when you add the coverslip after washing , try not to press it hard. just drop it from your fingers onto the LMPA. 

Third , what is expected to be seen on your slides when looking through the eyepiece of the epifluorescent attachment is green comets on a black background if your all work went well. was this what you saw at readable areas you mentioned ?  

Forth, if you are sure that you did well with the steps previous to microscopic visualization, then you should be sure that you are using the epifluorescent attachment the suitable way . i.e . , use the filter cube which is specific to your stain, close the fieldlight and just switch the epifluorescent light on. and work in a dark ambient . 

Last, if attach some photos ,  its gonna be easier to troubleshoot your problem . 

Best wishes , and let me know if you nees more details .