Hello Everyone. I’m currently trying to amplify a specific gene that involves a WT and a alternatively spliced variant, and my PCR products expected are around 300 and 250 in Bp. Now everything worked (RT, PCR) and showed up on the gel, once. I tried replicating the results today and only the ladder is showing up, 3 times in a row. The only thing I changed was the kind of ladder used because the previous one ran out. what is going on???
Heres what I did:
melt 2% agarose gel in 50 mL 1X TAE until no flakes are visibly seen
cool solution for 10 minutes
add 5uL SYBR safe, and mix well
pour into gel castor and wait for 30 minutes for it to solidify
remove comb and load ladder and samples using the parafilm method
Samples = 10 uL + 2 uL loading dye,
ladder = 4uL water + 1uL ladder + 1uL loading dye
run gel at 120V for 80 minutes
reagents:
1XTAE
NEB loading dye 6X
100bp DNA ladder NEB
SYBR safe
I also attached a picture of the gel that worked.
The fact that you see nice district, bright bands on your ladder means that the gel electrophoresis worked properly (no issues with the dye, the electrodes, etc.).
That tells me that the issue was your PCR (or RT-PCR) reactions. What did your positive controls look like?
Try making fresh working solutions of the components you are using in your PCR reaction and then check it on the gel.
Did you re-run your PCR, or did you just load leftover of your original PCR? If you're running the exact same PCR products, it's possible they broke down in the interim.
Also be aware that the more UV your labeled products or exposed to, the less bright your bands will be (although that should also affect the DNA ladder).
Can you attach an image of the gel that did NOT work?
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