I've been having issues with my mouse genotyping results. I normally make a 1.5% Agarose Gel and add Ethidium Bromide to the solution to be able to image the results. I let all my samples run for about 40 min at 120V. After viewing my images, the whole bottom half of my gel is not even visible. if you click on the photo, you can see that I have a second row of wells with replicates of the first row of samples. I can see the ladders and the samples in all the top wells; however, the entire bottom row of wells including the ladders are not visible at all. I am starting to think that there is an issue with how I made the gel.
Please help.
Bonjour Lucy,
Je voudrais savoir ceci: quelle est la taille de votre marqueur et des amplicons?
Hello Lucy Carrillo . How are you doing?
Honestly, there is nothing wrong with your gel for me. What is the size of the ladder your are using? The ladder is your guide in the gel. The last band in the ladder, may correspond to minor size of DNA band. The strong ones are references depending on the ladder you are using, sometimes are 500bp or 800bp.
Well, supposing that your ladder is for 100bp, in the gel, t the last band at 100bp, the above band should be 200, 300, 400, and 500, respectively. Your amplicons are between the 200 and 300bp.
So, if your desired amplicon from your primers amplified an 250bp DNA fragment, your gel are OK.
Another suggestion you can reduce the size of the gel to not waste reagents.
Good luck.
Hi,
@Tulio Teruo Yoshinaga @Inkale Basele CaGod
Thank you for your responses. I used a 1kb DNA ladder.
I understand that the top row looks okay, but if you click on the photo you can see that I have some wells in the middle of the gel as that I loaded with the same exact samples that I used in first set of wells. Yet, in the image there appears to be no bands at all. You can't even see the ladders that I loaded in the second row. I am unsure what the cause of this is. Why are replicates in my second row including the DNA ladder not visible?
Hi, Lucy Carrillo . Oh... I thoght that wells in the middle of the gel were empty. So did you put samples in there and run the gel.
Hum... Maybe it a problem during the gel preparation. If the EtBr were not well mixed witht the aragose.
Is this the first time that happen or you saw this before? If you prepared two small gels might resolve the problem.
Tulio Teruo Yoshinaga Yes, I apologize for not specifying that.
I ran the gel three times. I used different samples on my third run, but even then I still experienced the same thing where my bottom wells did not include any bands.
Perhaps, it is the EtBr. Maybe it is not being mixed properly? I also read online that the EtBr migrates towards the negative electrode. However, I didn't think that the gel was running long enough for that to be the case...
Lucy Carrillo Yes, probably it is a problem with the EtBR during the gel preparation.
This gel is a very big one, try to run in a small one. Once I ran a gel with EtBR, I beleive was 15cm by 15cm with three lanes of wells and worked fine.
I think a small gel will work as well.
You can also try by casting a 0.8% agarose gel in TBS.
The percentage of the agarose gel that you use for separating DNA PCR products seems to be too high.
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