Am using an ~ 6 years old Primers now, and am not able to get any amplicons, can this be due to degradation of nucleotides over time ? .
it depends on the storage conditions.....if still freeze dried they will last for ever but yours are already dissolved so it depends on the buffer and the temperature. If stored at high concentration in TE they will store for year at -20s but if stored dissolved in water ( which becomes acidic with time) and at low concentration ( absorbs on to container walls and lacks buffering capacity) then they will not last. Storage at -80 is better than at -20 ( which is still very good) but storage at frdge temperatures or above will shorten the storage life
Dear Manohar Raju
Pls. find the attached files may help you
http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
https://www.idtdna.com/pages/decoded/decoded-articles/ask-alex/decoded/2012/01/10/what-characteristics-make-up-the-most-efficient-pcr-primers-
http://www.ncbi.nlm.nih.gov/pubmed/11348798
Regards
Prof. Houda Kawas
Definitely. That's why I don't use primers older than 3 years.
It could also be absorption into tube walls - you can check that by re-measuring primer concentration.
At any case - it would be cheaper to get new primers and don't waste on enzymes in failing reactions.
it depends on the storage conditions.....if still freeze dried they will last for ever but yours are already dissolved so it depends on the buffer and the temperature. If stored at high concentration in TE they will store for year at -20s but if stored dissolved in water ( which becomes acidic with time) and at low concentration ( absorbs on to container walls and lacks buffering capacity) then they will not last. Storage at -80 is better than at -20 ( which is still very good) but storage at frdge temperatures or above will shorten the storage life
Asta Jokubauskaite, How do i check for remeasuring Primer concentration , is there a better way to do it .
Paul Rutland, Exactly Sir , but the manufacturer insist us to use molecular grade water to dissolve the primer , Does TE can affect the Primer ? , is there any concentration of TE to be maintained with the Primer ?
I use NanoDrop or Qubit, most organisations have at least one of them.
hello Manohar,
I have always used 10mM TE for resuspension ( see IDT attached file section 4) and all of the companies that I have used ( and many oligos that I made for my institute over a 5 year period ) were suspended in TE. The EDTA removes magnesium so nucleases are inactivated and the Tris buffers the oligo at alkaline pH so avoids acid depurination which takes place when the water absorbs CO2 from the air and forms carbonic acid which is destructive to single stranded DNA. All of the companies that I have used for oligo production recommend TE not water for routine use of oligos . Further dilution of the Te when adding primer to the pcr mix means that the effect of edta or tris in a pcr reaction is negligeable. There may be some specialist reasons to dissolve in water such as further binding of oligos to solid matrices but these are uncommon. In my opinion the higher the level of purification of the oligo the more important it is to dissolve in TE not water but I would always use TE
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