Hello, I recently purchased some primers from IDT (20 bp each), we got some problems getting the PCR reaction going, so we tested the integrity of the primers in an agarose gel. We see very different intensities on the signal of each primer. We used 1ul of stock solution (100uM) for each primer, immediatly after hidratation of the stocks on TE buffer (tris 10mM ph 8.0 EDTA 0,1mM). Is this common or our primers are degraded? It's not the first time we see these results on pure primers ¿can you tell me what should be the expected result? Best Regards
That looks about right for an agarose gel. However, short oligonucleotides are very hard to be visualized accurately on normal agarose gel. It has to be run on polyacrylamide gel, the type used for DNA sequencing. Just Google DNA polyacrylamide gel electrophoresis protocol or any other variations and you'll get the instructions on how to do it if you want to be absolutely sure about your primers.
it is a possible result as the single stranded oligos are short and do not intercalate much ethidium bromide so look very faint. If one primer can hairpin to form double stranded dna then it will have a much brighter fluorescence. The primers are very short so there may also be an effect of some sequences trapping less ETBR just because they are rich in a particular base
Hi Nicolas,
After looking more carefully at your photo, I'm concerned about lane 2, which is your positive control? I cannot see any band there, so either you diluted it too much, or there is some problem with your detection system, which could explain why you are not seeing your products. Is that chemiluminescence with SYBR Green? When I use EtBr gel, a 5 ul ladder load usually yields a brighter band than that.
We also use primers from IDT and in my experience it is very rare that they sell bad quality primers. What troubleshooting steps have you taken? How large is your product?
Best,
Thank you for your answers.
As for the second lane, it is very diluted DNA and it wasn't used in PCR, so we have no concerns with that matter.
The detection is made with a non toxic stain called SafeView. It usually gives higher signal, but this gel in particular was stored for a couple of days, so it lost a bit of the stain. We thought that the secondary structures in the primers could be changing the signal, but the difference looks so big that we weren't sure if it was correct, hence the question if it was degraded. Our product should be 164 bp.
We are now starting different aproaches to achieve the amplification, considering that the primers should be fine.
Thank you again,
Best regards.
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