hi guys
Does anyone know why my leader runs Crooked?
Conditions are TBE, 1% agarose, run at 85V for about 30 minutes and was added EtBr before running it. 1 kb ladder.
I would really thank you for your attention
You have not mentioned what is your sample and how it was isolated or purified. Nevertheless, I have a feeling that the sample is heavily contaminated with RNA. I can't think of anything else that would run this low and as a blob in 1% agarose gel.
Hi Nargess,
Try leaving a well open between your ladder and samples. It could be salt or other organic contaminants in the sample that causes the DNA in the well to distort, affecting your ladder's migration.
It seemed to be that your agarose gel might be problem (Did you check carefully TBE buffer or agarose was boiled completely before pouring in a tank?And did you store this gel for long time?). The DNA ladder looked like a strange migration even though it could be separated. Try to make a new gel again and load only your DNA ladder first (2 ul is enough amount), if it works well then you load your DNA samples together and compare.
Hope you get luck in your next experiment!
it looks to me like the agarose formed a blob on melting with insufficient mixing so you have a local region of higher percentage agarose that the sample and ladder are migrating round rather than through. If you still have the gel run the same samples jn different positions and it may not happen again. Swirl/mix you melting agarose more thoroughly. Leaving a space between the samples as suggested above is also good practise in case different salt concentrations in the ladder and samples has an adverse effect on how thay run
Hi Nargess,
I recently optimized an agarose gel with almost similar specifications.
1. Gel preparation: I prepared a 1% agarose gel. You may refer to Gene Cloning by Sambrook for selecting appropriate gel percentage for your DNA based on the number of base pairs. I think your gel was disrupted during the process of solidification. Incidentally, non-uniform solidification occurred and the density of the gel in different areas differ. It may be the reason why the DNA has taken a twisted path. In addition, you should also make sure that platinum wire of anode and cathode are present in throughout the length of the apparatus. If not, it leads to changes in direction of DNA migration.
2. Voltage applied: I usually run the gel at 4-5V/cm. You should calculate the distance between the electrodes, not the length of the gel. For example, if the distance between the electrodes is 15 cm, I would run the gel at 5*15 = 75 V. I run it for around one and a half hour (patience is precious) and I have generated band separation of the 1kb DNA marker just like they have shown in their manual. Its Fermentos 1kb DNA marker in my case.
3. Buffer & EtBr: I used TAE Buffer 1x. You should add EtBr into the gel and into the buffer. EtBr usually migrates in the opposite direction of DNA. So you may add it at the positive end of the apparatus.
4. DNA Miniprep: I usually use an invitrogen miniprep kit. but I have done minipreps using traditional methods and I am sure that you can achieve superior purity using traditional methods.
Please refer to Gene Cloning by Sambrook & Russel - Volume 1, Chapter 5. Protocol 1. It has all the information for tweaking an agarose gel.
This may be due to gel and buffer are stored for long time, change these 2 and use sterile autoclave water in experiment, wear gloves, reduce your contamination.
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