Full disclosure: This was the third run using the same gel and running buffer (I added distilled water to compensate for evaporation). I know you're not supposed to reuse running buffer this much, but this still doesn't make any sense to me, so I'd appreciate an explanation. Also, I'm a complete rookie so please give whatever advice you think is useful.
This was an hour run testing two fluorescent stains loaded with two DNA ladders. I did this because no bands were seen in prior runs from either ladder (PCRs were visible, but not ladders) when one of the stains was mixed into the liquid agarose. At around 15mins, I checked for bands under UV and saw DNA smears moving toward the cathode (3 tracking dyes moved as expected and the leads were definitely connected correctly). At 25mins, I took a second photo then flipped the orientation of the gel to double check that it really was migrating backwards. 10 minutes later the smears moved back over the wells and by 50mins they had clearly continued into the gel. Below is a set of general points and some photos in time series. Ironically, the old stain that wasn't working is the one we're looking at and the newer stain (SafeRed Loading Dye) is completely invisible. How did DNA acquire a positive charge? And why did the new stain not work?
UPDATE: I ran PCR products and a DNA ladder in fresh buffer and the ladder moved as we'd expect but the PCRs migrated backwards; tracking dyes moved normally. I've been putting 13uL 1M magnesium sulfate instead of water in my 50uL PCRs and I think that might be the problem. Still, I can't explain what exactly happened and I'll test this soon.
- Voltage was set at 60V and the gel is about 6.5cm
- 2% agarose gel was made with 1X TAE
- pH dropped from 9 to 8 throughout the run
- Current climbed from about 70 to 84mA
- The first lane was not loaded with anything and the last lane was loaded with a mix of everything + glycerol solution.
Yazen Alnefeesi Are you really sure that your connection (to the power supply) is correct: + to + and - to -?
Yes, I'm certain. Also, even if I wasn't sure the tracking dyes moved from cathode to anode just as expected. Here's a photo from before the flip. I also saw the tracking dyes backtrack after I flipped the gel.
I'm starting to think that this dye may have acquired a positive charge somehow. And since the DNA was saturated with it, the net charge of the dye-DNA complex became positive. Thoughts anyone?
did you thoroughly mix the running buffer between runs?
I am thinking that in an early run the buffer has become polarised by ions moving to the opposite electrode so we have a pH gradient across the length of the buffer chamber. Dna will have a net charge depending on the acidity of the environment it is in so at ph8.4 dna will have a positive charge and move as expected. If the end of the gel and buffer near the samples is ,for example, ph 10 then the dna will be negatively charged compared to its environment and will move towards the anode. The coloured markers are not dna and although the same logic applies they may just be partially changed in terms of charge so are probably moving more slowly but still in the right direction. The increase in current may be that the buffer is too dilute and much of the salt has polarised to the electrodes leaving a high resistance ,low salt region in the middle of the gel/buffer
I think this paper explains why my DNA migrated backwards: https://www.nature.com/articles/srep38628 Apparently, charge inversion happens when multivalent cations (possibly carried over from lysates) are present in a solution of low dielectric constant. The leading hypothesis is that these 3+/4+ ions were dragging DNA in the wrong direction after associating with the phosphates. Fresh buffer and gel seem to solve the issue, but my bands still become increasingly blurry throughout the run.
Would phenol:chloroforming the PCR product help with sharper bands? I'm thinking that some stalled Taq might be slowing mobility in some molecules and not others. Is that likely?
If you think that some protein is still attached to your pcr product just add SDS to make 0.5%sds to dissociate the protein and then load the gel. It is not usually necessary but does happen in restriction digest sometimes and is much easier that phenol chloroform. If your pcr bands are wiude and blurry then you may be running too many cycles or just running the gel at too high a current and the gel is heating up and the samples are spreading in the over hot gel.Try running the gel slower/lower voltage
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