To make a typical 100 microMolar (100X) stock concentration of primers, dissolve the primers in a volume of sterile distilled water that is 10X the amount of nmoles in the tube, using microliters of water. This value is printed on the side of the tube. For example, if your tube contains 53.4 nanomoles of primer, then you would dissolve using 534 microliters of water. This will now be at a 100 uM concentration.

then Dilute this stock 1:10, to give a concentration of 10 uM. store in -20 degree..

To make a typical 100 microMolar (100X) stock concentration of primers, dissolve the primers in a volume of sterile distilled water that is 10X the amount of nmoles in the tube, using microliters of water. This value is printed on the side of the tube. For example, if your tube contains 53.4 nanomoles of primer, then you would dissolve using 534 microliters of water. This will now be at a 100 uM concentration.

To prepare primers for use, Dilute this stock 1:10, to give a concentration of 10 uM.

It is true that every synthesis will have a different yield, whether from the same company or a different one! When it comes to primers, I take different approaches based on the downstream applications-

1. If I need an approximate concentration of primer, I use the amount of primer that the company indicated in the paperwork and dissolve in enough water or TE (10 mM Tris pH 8, 1 mM EDTA) to make either a 1 mM or 100 uM stock. For instance, sequencing often wants everything in water so as not to interfere with the reaction, but the primer may last longer in a buffered solution if you will add it to PCR. From this stock, I then make further dilutions - to be accurate, if I need more than a 10 fold dilution, I create serial dilutions. It never hurts to make aliquots so you have less freeze/thawing.

2. If I need an extremely exact concentration, I would aim to be MORE concentrated in the initial resuspension and then measure the actual concentration on a spectrophotometer. Sometimes the number from the company is significantly off, but usually it is pretty good. Calculate the concentration based on the extinction coefficient calculated for the primer based on sequence as nucleotide bias in small primers can significantly effect this value. Dilute as above.

3. Ack? Why doesn't my primer work??? There are two main possibilities that I usually run into - salt or weird organic stuff leftover from synthesis and deprotection or a bad synthesis. To get rid of these problems you can use a desalting column (this will get rid of weird salt or organic stuff), gel purify (this will get rid of weird salt or organic stuff AND show you if the synthesis gave the right product), or purify by HPLC (this will get rid of weird salt or organic stuff AND show you if the synthesis gave the right product). Gel purification and HPLC also let you purify a correct product away from an incorrect synthesis product.

Because our primers are typically used for either PCR or sequencing, I prefer that both stock and diluted aliquots are in water. However, I also know that some researchers prefer TE for long term storage. Generally I would recommend that the stock primer solution be diluted in TE and the diluted aliquots be diluted in water. TE can interfere with applications like sequencing and PCR.

go as said by Holger Fey ...he is right..but dilute primer in MQ water. from main stock you can further dilute it to pmol/ul e.g if main stock is in nmol/ul diluting it 1:10 makes it 100pmol/ul further diluting it to 1:10 makes it 10pmol/ul and so on...

To make a typical 100 microMolar (100X) stock concentration of primers, dissolve the primers in a volume of sterile distilled water that is 10X the amount of nmoles in the tube, using microliters of water. This value is printed on the side of the tube. For example, if your tube contains 53.4 nanomoles of primer, then you would dissolve using 534 microliters of water. This will now be at a 100 uM concentration.

then Dilute this stock 1:10, to give a concentration of 10 uM. store in -20 degree..

Stock solution: nanomoles of primer X10 (using water for injections without NaCl).

Working solution: 9 parts water (as above) and 1 part stock

This way you should have the same primer concentration in a clean sterilized solution.

the information about the volume of water you can find on the information sheet which comes with your primer tube. dissolve the primer in the indicated volume (MQ water). add MQ water, vortex very good, wait 10 min at RT, vortex again. than you have a 10x stock solution. for usage prepare a 1x aliquot by mixing 9 parts MQ water with 1 part of your stock.

A calculation for making a master stock and working stock is attached hereby.

I generally make 100 pmol master and 20 pmol working stock of primers. I dissolve it in sterile milli-Q water by add 65° C warm water and incubating in water bath at 65° C temp for 10-15 min with occasionally mixing. In my hand it works fine. But most of people dissolve prime in 10 mM TE.

I generally make 100mM by multiplying with 10 of  yield given in primer's document and its master stock. For working I am using 10mM and it is working nicely. 

Dear Hamza,

 We syntesis primers from Eurofins. When you ordered the primer you might get a sheet with primer detais. If you observe carefully you will see various amount mentioning yield ug/ul, yield ng/ ul etc. below that column you will see a digit like 47.0 or 307 etc. what you have to do if you want your original stock in ng/ul just add that much ul of MQ in it then it will be of that much conc. e.g. if you see yield ng/ul column with 47.0 then add that much MQ to itthen your original stock will be in ng/ul i.e 1000 pmol/ul. 1ng=1000pmol.then make a stock solution from it just take 10ul of original stock and add it to 90ul of MQ Then it will be 100pmol/ul. from this make working concentration we use 10pmol/ul for semi quantitative PCR. For 10pmol/ul what you do take 10 ul from 100pmol stock solution and add it to 90ul MQ so you have 100ul of 10pmol/ul primer.

Note- While dissolving the primer first centrifuge the tube then add MQ. mix it properly or vertex for 15 sec again centrifuge. then proceed for other dilution.

Hope you might get what i told. All the best.

http://webserver.mbi.ufl.edu/~rowland/protocols/PCRprimers.pdf

I hope it will help

Things will work along with the  instructions on the sheet provided with primers.

Resuspending PCR Primers

Materials

1.       ‣Molecular Grade H2O

2.      ‣Primers (dry)

3.      ‣Sterile Microcentrifuge Tubes

Method

Primers are often shipped and received in a lyophilized state. First create a master 100 × stock (for each primer and then dilute it to a 10× working stock.

This reduces the number of freeze/thaw cycles that the master primer stock goes through and reduces the chances of contaminating the primary source for the primer. 

Spin Down Tubes

Primers should always be spun down before opening the tube for the first time.  The pellet can often come dislodged during shipping and may be in the cap!

Master stock, 100 µM 

100 µM = X nmoles lyophilized primer + (X × 10 µl molecular grade H2O) 

To determine the amount of H2O to add to the lyophilized primer simply multiply the number of nmol of primer in the tube by 10 and that will be the amount of H2O to add to make a 100 µM primer stock. 

For example, if there are 25 nmol of primer then by adding 250 µl of H2O, a 100 µM primer stock is created.

The original primer tubes are often used for this 100 µM stock.

Master stock primers newly suspended in H2O should be allowed to sit at room temperature for 10 minutes before they are used for working stock dilutions.  Mix well before making working stock dilutions.

Working stock, 10 µM

Dilute the primer master stock in a sterile microcentrifuge tube 1:10 with molecular grade H2O. 

Best Regards, 

M, Roshdi

Excellent

Excellent .thanks 

Excellent Mehdi Roshdi Maleki

Along with primer one sheet is provided by manufacturing company and give the detail of how much ul of MQ water to dissolve the primer to make 100umol stock