whenever i buy new primers (100pM) they work fine at first dilution (10pM) as well as first 3-4 polymerase chain reactions. But when i use same or new dilutions (10pM) after 15- 20 days at the same annealing temperature or Gradient used earlier, amplification fails all the time. What can be possible reason for that; if primer contamination then how can i purify them manually.?
If you are dissokving the primers in water then absorption of CO2 from air makes it acidic and depurination occurs. Dissolve them in TE. The tris buffers alkaline and the edta removes divalent ions so any nucleases present cannot work
Hi Yaser,
One thing you need to rule out is primer degradation. This could be due to contamination introduced by the water used to dissolve/dilute the primers, the pipette tips, the eppendorf tubes, etc. Autoclave everything and make sure to keep them sterile.
Keep in mind that while everything (water, tips, tubes, etc) is sterile and good the moment you open a new package or right after autoclave, once you start using them, how clean they remain all depends on how they are being used.
Hope it helps. Good luck!
Best,
Sheng
If you are dissokving the primers in water then absorption of CO2 from air makes it acidic and depurination occurs. Dissolve them in TE. The tris buffers alkaline and the edta removes divalent ions so any nucleases present cannot work
Yaser Rafiq Mir What type of water do you use for primers dilution? How do you store the diuted primers? I met once such problem with MQ water stayed in the system for a few days without use (after holidays).
this is actually a great question. A really good suggestion was to NOT use water for storing primers, especially stored at low (working concentrations). Water can be slightly "acidic" in some places, if you are not buying water. And indeed water + CO2 generates carbonic acid. Gradual loss of primer specificity is good indicator of degradation of oligos. We assume that the solutions are DNase-free. Suggestion of adding TE addresses potential presence of DNase coming from fungi/bacteria or misc other sources like tips. However, even if traces of DNase are present, it should not impact your oligos if you keep your oligos on ice and assemble your PCR on ice (not everyone does). The fact that the loss of activity happens gradually is a good sign of "acidic" water ruining your oligos. in the old days some 30 years ago i used 10mM tris (not EDTA) to dilute stocks of oligos and especially "working concentration" oligos. This is to avoid bringing EDTA into sensitive PCR reactions, which critically depend on concentration of ions. EDTA is a chelating agent. If you are really really concerned about DNase in your reagents then diluting stocks in TE is ok but i do recommend diluting working concentration (10-20microM) of primers in 10mM tris HCl, ph8 to 8.5. This is to avoid EDTA carryover into your sensitive PCR reactions. sometimes the volume of your primer may be 5-10% or more of the volume of PCR reaction so if you have 1mM EDTA in your primer solution, you bring 0.1mM EDTA into your PCR reaction (10% of 1mM). A helpful step to address concern about degradation (also to increase specificity) is to assemble your PCR on ice, add enzyme as a last step, and then use "hot start" PCR method (heat up your cycler to 94 or 95C and hold, put your tubes, close the lid and continue cycles. This guarantees that DNase (if any is present) will be inactivated, and also increases specificity (less unspecific annealing, and no concerns about chew-back activity, if you are using Exo+ polymerase. This is a fun question to answer, and following these guidelines does help a lot with improving reproducibility, specificity and yield
May be your primer got degraded. Prepare it freshly and start your PCR
I suggest you to dissolving your stock primers (100pm) with TE buffer since it is more suitable for longer storage. However, you can just use sterile water to dilute your stock primers into your working primers (usually 10pm). Still, if you have enough TE buffer, there should be no problem for you to dilute your working primers by using TE buffer.
Make smaller aliquots of your primers, don't leave them on your bench all day, store them in the refrigerator for short term (~2 week) use, and DO NOT freeze and thaw multiple times. Primer degradation is common.
Classic problem, easy solution.
I have used MQ water, autoclaved distilled water, double distilled water and Sterile water (supplied with injections) for primer reconstitution and dilution. I store working primers at 4 degree centigrade@ Alex Ignatov
Thanks for guiding @ Paul Rutland , Sheng Sun , Alex Ignatov , Igor Nasonkin , Shayanki Lahiri , Norhidayah Rosman , Katie A Burnette
Dissolve the primers in T.E buffer and maintains its pH to normal, if pH changes, it may be water or T.E buffer the Polymerase Chain Reaction will not be properly proceeded. One more thing, Autoclave your tubes in order to finish the contaminants and then use then it will also enhances the reaction towards perfection.
what concentration of TE should i use to dissolve primers. I mean 1X or 10X @Paul Rutland
1x TE is strong enough for primer stability and will not have any effect on the buffering capacity of the pcr buffer. If you use 10X then there is some risk of removing Mg ions which are essential for the pcr reaction to work Yaser Rafiq Mir .
Yaser Rafiq Mir ,
I use PCR grade water all the time to dilute my primers to 20uM. You mention that you are using 10 pM which is very little. Are you thawing your primers fully, vortexing and spinning down (Since the concentration is very low)? Are you running a positive and a negative control in your PCR reaction? These might help you in your PCR reaction.
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