I performed a reverse PCR from a cDNA plasmid template in order to skip one exon of my gene of interest. Product size seems the correct one (5.7 Kb). But I don't know the reason why the bands on the gel look that weird. Agarose gel concentration is 0.8 %. The idea is to cut out the band and then do a ligation of the linear DNA PCR product. This funny shape could be a problem for such a porpouse? Any idea of what's wrong?
Thanks
It hapen when the run is fast .slow dow the running .and you have too much DNA on the slot.
You did not mencion the voltage.
I agree with both Maria and Aliakbar.
You may want to decrease the running voltage for your gel, especially when aiming to obtain products that are small in size as this might be problematic for downstream purposes. Although it may take a longer time for your gel to run, you will have lesser risk of getting curved and distorted bands which are indicative of your voltage being too high. You can also use a freshly made TBE buffer to dissolve your agarose and also as your running buffer.
Best of luck.
HI Ivan, All three answers above are correct & can help solve your problem to some extent. Reducing the running voltage will minimize the streaking you see at the top of the gel. Loading less per well will also help. Making sure the buffer composition in the gel & the running buffer is the same & fresh buffer would help. But a bigger issue I see is if this is a PCR product that you are running on a 0.8% gel, and the template DNA is a plasmid containing a cDNA clone - why do you have other bands present in the gel? That to me indicates you either do not have optimal primer design or optimal PCR conditions. If the end goal is to clone that PCR product into something else you need to optimize that step 1st. Answering your other question if the shape of the band on the gel would be an issue in down stream applications - the answer is no, as long as you have a clean PCR product.
I have experienced this type of problem before due to overloading of the product in one well. Check your primer specificity also.
Hope for the best.
The problem might be due to non-uniform solidification of the agarose gel. Make sure to completely dissolve the agarose in buffer before casting.
Use fresh TBE buffer for dissolving agarose as well as for running gel buffer.
The problem might also be due to increased voltage/current. Try to reduce the voltage.
I agree with Wasim, most of that were the reasons for similar gel pictures I had sometimes. I've found that specificity of primers and PCR cycle conditions are often affect the bands you see. I've also been taught (when it is critical!) to start gel running at low voltage first (for a few minutes) to allow DNA enter the gel, and then you can increase the voltage and run gel fast - that actually helps and you might have a nice gel picture! And yes, you can cut the band you need for next manipulations - you should be fine. Hope it helps!
I think that the uneven running of the samples is probably caused by too much salt in the dna samples . Try desalting or running less sample and as mentioned above run at a lower voltage
It could be a different story, but I received similar picture after running ligation reaction on gel. So the answer form Paul Rutland could be correct.
Lots of answer already. And most of them are possible factor behind this problem. And my recommendation is step wise troubleshooting. 1. Primer and PCR condition optimization according to your reference data. 2. Freshly prepared buffer and gel uniformity. 3. Clean electrophoresis chamber and use same fresh buffer 4. Low voltage run.
Hi, I also met this kind of problem when I run 2kb DNA sample, 1. Check your primer specificity and PCR condition. 2. What kind of enzyme did you use（does your enzyme high-fidelity thermostable or not) ? 3. Try to cut out and sequence the band. 4. Run at voltage 100V for at least 40 min. 5. Your template quality and concentration also play main role for successful amplification.
When I got this kind of band and sequenced it, I found that some of the nucleotides were missed.
Best luck
Hi Ivan, Few things are very important in agarose gel, Concentration of amplification product, PH of the running buffer and gel making buffer, and Voltage. According to your gel picture, Your DNA marker separated well so there is no problem in your gel preparation, PH of the running buffer and voltage. But your amplification product loaded well only weird. Because PCR product load is high. Just you can load less volume or reduce the concentration cDNA for PCR amplification. And your DNA marker band also not sharp if you need you can use 1% agarose gel.
All the best..
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