The first lane is DNA ladder, I persume that the problem is too much loading of ladder in this lane or the electrophoresis time is too short. The second lane is a primer I designed for sul1 gene, the third lane is also a self-desinged primer, the third one is from the liternature. From this result, may I persume that the first primer is fail because it looks fading? Then the second one have one band around 100bp, the third one had 4-5 bands?
I use 1% agarose gel and the first picture is 120V 20min, the second one is 110V 40min
What should I do to make it look better? I also want to send this sample to test the sequence, is it ok just to cut it down?
Also how can I use this result to quantify the gene?
Thank you so much for reading this. I have little experience in biological experiment I just getting started....
Dear Chloe Park,
You are correct. As I can see in the image, the band is very fainted in the second lane, indicating that the primer is degraded.
The degradation is due to improper storage of the primers. So, I suggest you always use aliquots rather than the stock solution of the primers.
Keep the primer stock in the deep freezer (-20 C), and do not frequently freeze-thaw the stock solution.
You can rerun this primer on the gel electrophoresis for at least 60 minutes. You can also use the freshly prepared TAE buffer. Following these instructions, if the band is not intact, synthesize fresh oligonucleotides.
Best,
Samrendra
Hello Chloe Park
Try using thicker gel for such a small fragments (e.g. 2% or even 3%). Use less ladder (red color indicates saturation); First lane really looks like primer-dimers, maybe PCR conditions were not right (lower annealing temperature); in second, especilly third lane you have unspecfic primer binding, try higher annealing temperature, less primers...
All in all you should optimize PCR conditions.
Good luck!
What size PCR product(s) are you expecting? You should know that before you set up any PCR reactions.
PCR products less than 100 bases in length should be cloned into a plasmid before you try to get them sequenced. The "messy" part of a sequence read makes it hard to get data for the first 30-50 nucleotides.
No, you cannot quantify gene expression using standard PCR from genomic DNA.
I'd recommend you show these results to your mentor and talk about your project before you do any more benchwork.
Thank you so much for replying!!!!!!! Marina Korolija Katie A S Burnette
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