For carrying out an Amplified Ribosomal DNA Restriction Anaysis (ARDRA) I had to separate the products of restriction-enzyme-digested cDNA in an agarose gel. The agarose concentration was 1% (w/v) in 50 mL 1x TAE buffer. I used 3 µL Midori Green Advanced DNA dye and 1 x loading dye. After the separation process, the gel showed weird formations. I can't explain myself where they come from and what they are. Sadly the gel broke when I removed it from the chamber, so that's why there are two white horizontal formations. On the right the DNA ladder standard.
Has anyone seen this before and can explain what happened?
Thank you!
MF
I have never seen this effect before but the gel looks like it is running well since the ladder has separated properly. My guess is that you picked up the gel with bare hands or dirty gloves and the pattern is finger marks on the gel surface. The lack of any digested dna pattern in the gel may be caused by loading too little dna but this is very much a guess. An alternative is that the gel wells were split when removing the comb and the smears are your dna samples running underneath the gel and mixing but not running through the gel matrix although you should have noticed this leakage while loading the gel. I am assuming that when you say loading buffer 1x that this refers to the final concentration of loading buffer just prior to sample loading. Too little loading mix could make the sample too light and allow it to escape from the wells before the run starts. Again if this was the case then you should have seen sample spreading out of the top of the gel well during loading
All possible reasons are already mentioned earlier.
But I just wanted to mention you or anyone who might be following/looking for similar problem.
- Self trouble shooting before abruptly asking for help
- Think, did I see this kind of result before in my own experience?
- As you call "wierd formation", is it in gel or outside gel. If one can not say what it is (however Paul Rutland already said they are fingerprints), it is absolutely going beyond the lowest limit of the respective gel. So, it must not be the thing which is cause from inside the gel. It is something external factor.
- Why not I just repeat the same process with more care and see what went wrong in the previous gel (usually people do not use the complete PCR reaction for gel). If there was nothing on the gel, its obviously useless to proceed forward or save the PCR product.
- Is it very first gel, did I verified the protocol before proceeding to the actual experiment?
- If the gel is already broken/destroyed (looks like you dropped the gel, and its dust all over the gel), it is not suitable to present or ask question related to it. Better to just repeat.
Apart from these suggestion I was wondering what you are actually doing and why you are doing in the way you are doing. It consists of two section technical and method based
- why are you using 1 % gel, for better separation, you can increase your gel concentration and take the minimum possible gel piece with desired DNA band
- why do you use 1x loading dye? Generally 6x loading dye is used in 1/6 concentration of loading volume
- Did you get any product/bands in any previous similar gel? Or is it just very first gel?
- Midori Green is non-carcinogenic, still it is required to use gloves
2. Method
- What is your aim, why are you doing ARDRA? What is your gene?
- Why are you using cDNA? how did you get it?
- What protocol are you following? Who gave it to you?
As far as I understood the method, it is a version of TRFLP/-RFLP which uses amplified product of gene, i.e. DNA and not RNA. Maybe a look back at the aim and method looks relevant here.
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