I attach a photo of my agarose gel.. As you can see the DNA ladder runs like "all toghether" (sorry for bad english). I followed the manufacturer recomendations (1,5% agarose, 90mV, 1 hour)... The ladder goes from 100 to 1000 bp.. what am i doing wrong?
Just guessing..., is your ladder getting aged? As your product is quite small (say ~300 bp, assuming you are using a 100bp ladder) I would try improving resolution running a 2% agarose and shorter run times (say 20 min, for instance). 1 h is quite a long run, isn't it?
the ladder it's brand new... and yes i load 3 ul... i will try loading 1 ul! i hope it works!!
thaks!! :)
Agarose gel electrophoresis running and getting good band many factors are responsible
Like…
>>Making gel-
-how much % agarose depend on upon how much big or small DNA fragment have to separate
- You have to take same X buffer (1X or 0.5X) to make agarose gel *
- Proper melt and mix *
- Very important proper solidifying (give proper time to solidify) *
>>Sample preparation-
- DNA loading dye 6X, final concentration should not less than 1X
- Proper mixing
>>Placing gel into the tank
- Gel should fully dip with buffer
- Well front should be side of negative electrode , black cord
>>Loading-
- Load only into wells
- Try to load fast, because first well which you load it start diffusing
>>Running-
- It depends on area of gel, size
- Time also depends on upon minimum DNA fragment size and agarose gel size.
After seeing your gel, I have to say that you focus on suggestion which have star(*)
and use 1% agarose (1 g/100 ml)
And me too, sorry for bad English…
Thank you
all the very best
Hi Lino, just guessing~ First, you should make sure that every materials in this experiment are right ( such as the TAE bufferr). Maybe you should use 2 % gel instead of 1.5 %, if the size of your target DNA is around 300 bp; I think you should use around 90 V (60V~150V, just depent on how quickly you want to finish it) instead of 90 mV for 20 min; the gel you made may be a little thicker, 3~4 mm will be OK, and you should cut the gel cleanly before you run it; and the nucleic acid dye will affect the results too. Thanks. Good luck for you!
Definitely agree with Shuaipeng, 90V not mV. I overlooked that as a typo.
To achieve a good resolution agarose gel in TAE or TBE buffer, 10X are to be filtered on cellulose nitrate membrane 45μm. The cencentração gel should be 1.5% -2.0%.
I definitely agree with the above comments. To add up to them just make sure that the buffer you use in the gel tank is not very old too. Old buffer can cause poor resolution as well.
Dear Agustina Lino,
First : Boil the agarose properly, un-boiled agarose can make this faint resolution.As bands can pass through amorphous agarose. So, when DNA pass the un-boiled agarose retain some DNA that cause a dull band.
Second : Run in 250 volt for 20-25 minutes in 1.5% Gel. Longer run sometimes can causes such faint bands.
Third : Take gel photo as early as possible after the run completion. Making delay in imaging cause the band to diffuse through the gel, as there is no running force what we used (Electricity) to guide the DNA from Negative to Positive.
Good Luck !
i loaded 1 ul maintaining the running conditions, and it has improved a lot. I will try some of your other sugestions to keep improving until i get puclication quality pfotos THANKS!!!
2.5% agarose/1X TAE gel. Microwaved for 2' (with intermittent stops to prevent boiling over). Cool on a stir plate to ~55ºC (if it doesn't burn the inside of your wrist, it is OK to pour). Pour, and let it solidify (~5-10' depending on ambient temperature). Load 0.5 µl of ladder. Use half of your PCRs—you're definitely loading a lot. Depending on the size of your gel box, 150 V for 20' is fine. Stain for 20' in 200 ml of 1X TAE with 20 µl 10 mg/ml ethidium bromide (with gentle shaking or rocking). Wash briefly with water a few times, then wash with water for 20' with gentle shaking or rocking. And image. And your gel should look like this:
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