Hello - I am screening DNA extractions (all samples have a DNA concentration of above 100ng/uL) for Giardia intestinalis (genes gdh, tpi, and bg), and recently my gel is smearing. We haven't changed anything (reagents or conditions) from when it used to work. We make a 2% gel with 1x TBE buffer, use GelRed, let it cool for 30 minutes, and run it at 80V for 36 minutes. We also clean our apparatuses each time. I am confident in our PCR since we have not changed anything from when we started. So far, I have tried the following:
1) Cooling for 45 minutes
2) Running the gel at a lower voltage for longer
3) Running the gel at a higher voltage for shorter (picture below)
4) All new reagents - TBE buffer and ladder
5) Putting 5uL of DNA + loading dye into the wells
6) Putting less ladder into the wells: we started at 4uL and now we have been putting in 2uL.
I have attached a photo of our gels below. As you can see, they are quite streaky, and we are not able to accurately see our sample. In the gels, we have positive and negative controls, and we are testing the bg gene, which is 511bp.
Any suggestions on how to make our gels less streaky?
I have had similar problems with Gel Red which I do not see when I use ethidium bromide. It seems Gel Red itself can result in streaking (in my experience). I would suggest using far less Gel Red than instructed (no more than 1ul per 100ml gel) and much less ladder and PCR product than you have been using. I would not use more than 2ul of each. Try as little as you can and see if this resolves the sreaking issue.
- Agree with Craig about loading too high a mass of ladder and DNA
- the problem Craig describes is also exacerbated by too much DNA so if you load less you can - and we successively do - use the normal amount of gel red (although less will save you money admittedly)
- there might be another potential problem
- 2% gels should be boiled intermittentlay for 4-5 min with gentle swirling at the end otherwise you end with small agarose particles in your gel
- this can result in the sort of buckling and smearing you see as well
I use the 5 µl of DNA were separated on agarose gel electrophoresis using 2% (w/v) agarose in 0.5x TBE buffer. Electrophoresis was performed at 80 Volt with 0.5x TBE buffer as running buffer and then the gel was stained with 0.5µg/ cm3 (w/v) Ethidium Bromide solution.
Electrophoresis Buffers:
· 10x TBE buffer: is according to (Sambrook et al., 1989).
108g Tris-base
55g Boric acid
9.3g EDTA, pH 8
Made up to 1 liter with distilled H2O.
Dilute 50 ml stock solution with 950 ml d.H2O to obtain 0.5X concentration.
· Loading buffer (for DNA)
0.05% Xylene cyanol
0.05% Glycerol
0.01% Bromophenol blue
good luck
Hi all, thank you so much for your suggestions!
I continued with my 2% gel. However, I decreased the amount of ladder (3uL - I tried 2uL, but 3uL was better), DNA (2uL), and gel red (1uL - I tried 1uL per 100mL so I ended up getting 0.3uL, but 1uL looked better). I have been running the samples at 90V for 30 minutes. The streaking has improved, but the samples are still not as clear as I would like them to be.
I screened for two genes: bg and gdh. It seems gdh is much clearer than bg (seen in pictures below). Do y'll have any more ideas on how to make both bg and gdh samples clearer?
In the photos, I have marked the positive control with a red + sign.
Thanks!
Try on 1.5 % and even 1% of agarose, also less amount of DNA.
I think 36 min is not suitable for 2%, let it run longer
If you can make the amplification specific and efficient on the front end, that goes a looooonnng way to solving the problem on the back end (gel).
Upload any previous gel run pictures when it was working fine. Try to dilute your samples and then run on agarose.
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