Hi Alexandra...

I also agree with the above suggstion...however I usually dissolving the initial PCR primer stock in TE buffer (but I keep low conc of EDTA as compared to regular TE buffer)......subsequent dilutions for use ...are done in sterile water that further reduces the EDTA amount....Low amount of TE doesnot affect most of the regular applications...however for very fine and high end applications this maay not be desired..

I see one advantage.... the primers ...are less prone to degradation ...over a long period of time...

In sterile distilled water normally.

Alexandra, I share the Rachid suggestion.

I liked and agreed with Rachid suggestion. Only add here to check its composition/quotation list with primers for dilution factor.

Hi Alexandra...

I also agree with the above suggstion...however I usually dissolving the initial PCR primer stock in TE buffer (but I keep low conc of EDTA as compared to regular TE buffer)......subsequent dilutions for use ...are done in sterile water that further reduces the EDTA amount....Low amount of TE doesnot affect most of the regular applications...however for very fine and high end applications this maay not be desired..

I see one advantage.... the primers ...are less prone to degradation ...over a long period of time...

Hi Alexandra

Usually, the stock batch of the primers is meade in 100 PM. Please note the best concentration of primers to use in PCR is between 5 to 20. I usually use 10. For this purpose you may dilute it to 1/10 with TE buffer or DW. TE buffer is better for long period of time storage.

tx

Hi Alexandra

Usually, the stock batch of the primers is made in 100 PM. Please note the best concentration of primers to use in PCR is between 5 to 20. I usually use 10. For this purpose you may dilute it to 1/10 with TE buffer or DW. TE buffer is better for long period of time storage.

tx

The better dilution can be in DEPEC treated water, however the TE can be made also.

If you having problems to amplify measure target DNA carefully and try primers at 2 PM, and go increasing. Try also gradiente using TM of primes from 5 minus to 5 higher temperature of hibridization .

Ordinary primers goes in to pure MilliQ water and primers with fluorescent tail goes in TE. Never use TE for routine applications. For dilution please consult to lab technician.For PCR component calculation please refer to

http://www.biometra.de/1070.0.html and download the Excel tool for PCR and lets see what this can do for you.

1) Never expect confidence since your 30th PCR.

2) Do not focus on PCR but rather to your targeted sequences or fragments.

3) Try similar PCR conditions from alike articles of your topic (without dipping into strange adjutants).

4) Thousand researcher do thousand different freaks.

When the subject comes to the fish, salmon or trout DNA extraction and PCR conditions could be taken as example. Fish DNA (depending on the quality, from tissue or fin matters really!) could be amplified as any ordinary DNA. Well the main issue is the type of DNA (genomic or mtDNA?) may effect the PCR due to the size. I have not seen any difference between fish and other eukaryotic animal PCR conditions. This is not the exact place to lecture about this deep topic but just keep these thing in mind.

Thank you!

You are welcome

I have a question to everbody

what is the best way to get miRNA from peripheral bood ? Sample colected with EDTA or without any anticoagulant or preservative?

Mario

Well, DEPC water can be acidic which may cause the oligo to degrade. TE buffer (10mM Tris: 0.1mM EDTA; pH 8.0) is the safest to dilute primers. Be careful in preparing the TE buffer as the EDTA should be 0.1mM not 1mM which is used conventionally. Because if the concentration of EDTA is higher, it will chelate Mg- ions and inhibit the PCR.

Sometimes you might increase the concentration of MgCl2 0.5 or 1.0mM more which is 1.5mM normally in the provided buffer.

It is recommended to briefly centrifuge the tubes of dried oligo prior to opening them. This will ensure that the oligo pellet is at the bottom of the tube and will not be lost when the cap is opened. After adding water or buffer, briefly vortex the tube, but do not centrifuge it. Sometimes heating at 65 deg Celsius for 10 min might be required for complete dissolution of the primers.

Finally store the stock at -20 deg and avoid frequent freeze thawing.

Hi, I only dissolve PCR and FISH primers with milliQ water and I never have any problem with amplification and hybridization, High concentration of TE could affect ,check your primers and combination of them

Hi,

I think sterile water should be fine to dilute Primer. TE buffer may affect most regular application.

i generally use sterile water for primer dilution (my primers size will be around 17-22 bp) i never got problem, and i used it for more than 3 years there is very less negligible degradation after so long time.

liked and agreed with Asaduzzaman that TE buffer (10mM Tris: 0.1mM EDTA; pH 8.0) is the safest to dilute primers. Be careful in preparing the TE buffer as the EDTA should be 0.1mM not 1mM which is used conventionally. Because if the concentration of EDTA is higher, it will chelate Mg- ions and inhibit the PCR.

Hi,

I heard some opinion that TE buffer can potentially inhibit PCR reaction (polymerase) and sequencing performed with Sanger methods. I every time use the nuclease free-water to dissolving primers.

please can you tel me in wish volume of water am suppose to dilute this (10mM Tris: 0.1mM EDTA; pH 8.0) and obtain a finally solution of TE 1x

I use sterile water since TE buffer may affect most regular application

Its better to use HPLC grade water or commercially available PCR grade water. I have always used water.

I always dissolve PCR primers with double distilled water. Moreover, I always keep the ration of primer to water as 1:3 (1 primer, 3 water), and i obtain good amplification.

double distilled water is better