Primer dilution --> problem.. - (Jun/17/2011 )

Hello,

I've got a problem - received lyophilised primers and diluted to 100pmol/µL. Left them overnight in +4 C. Then i took 25µL of that solution and added to 975 µL H2O. So i have a final concentration of 2.5µM (working stock). Am i right?

Then i took 6.4 µL of the latter and added to my pcr mix (20µL volume) - to get final concentration of 0.8 µM. But the PCR failed.

Am i wrong in my calculations?

Thank You very much

i guess you over diluted your stockitself...100pmol per microlitre doesnt seem to be like stock...Another thing i would like to remind you that i think you totally missed a nanomolar concentration inbetween pmol and micromol, this makes further 1000 times difference from your req conc..

Thanks for your reply.
I ordered primers from metabion. Data sheet says "for 100 pmol/µL dissolve in (µl): 273". So I did that and got, what i call - stock concentration.
So i did another dilution. i've mentioned, that i need working concentration to be 2,5 µM. So i took 25µl from stock dilution and combined with 975 H2O -> so i assume that i have my working concentration of 2,5 µM.

For PCR applications for 20µL reaction mix i use 6,4 µL from working stock - so i get 0,8µM ?

Additional primer info:

5.2 OD
27,3 nmol
MW 5083

thanks

P.S. I'm asking you this, because I ordered new primers and I get smear all over the lanes. Also changed polymerase. And I'm using new dNTP mix. Before that i had positive result using old primer (which is, in fact, the same)by ISSR tech

now its clear to me, you have 27.3 nmol primer right?? so if you dilute your primer with 273 microlitres of water you get 100 Micromolar final conc. this will be your stock (so your dilution is right, may be i got confused with the pmol notation)...now you can make your working concentration 2.5 micromolar.

To get the working conc of 2.5 micromolar you have to take 2.5 microlitres from the stock + 97.5 H2O/T.E whatever you prefer.

And yes if you add 6.4 microlitre from the above working your final primer conc in 20 mcirolitre rxn would be 0.8 micro molar , but are you sure you have to use 0.8 micro molar of each primer (F and R)?. better have a check over it and proceed...

hope this helps.

GNANA on Fri Jun 17 20:09:30 2011 said:

Thanks for reply, GNANA. I don't use forward and reverse primers - for ISSR application only one primer is used in this case - the same for forward and reverse. So i would like it to be 0,8 µM in the mix. But the problems is that when i used it (got it, didn't dilute it myself) in the same concentration - the amplification was obvious and the patterns were clear. But now, when i've diluted it myself - all the lanes smear much - and almost no amplification. i am very curious what could happened.. going to lab tomorrow to make the things clear and if the cause of the problem will be found - i'll post it .)

thanks for your attention

We take primers from metabion. The stock solution is indeed 100pmol/µl = 100µM as written on the sheet.
Your calculations seem right, but I wouldn't dilute the primer that much and not to 1 ml, 2.5µM is pretty low concentration. I would personally dilute the stock 10x (like 5µl + 45 µl of buffered water) and use 1.6µl for the reaction, and prepare only 50 - 100 µl of the working concentration, depending how many samples you run at once. (but you may have reason why to use exactly 2.5µM, like pipetting larger volume, that is more precise or do hundred reactions at a time).
There may be problem with degradation of primers in low concentration, do you use water, TE or 10mM Tris pH8 for the dilutions?

Trof on Sat Jun 18 09:27:09 2011 said:

Thanks for Your reply. i use water and always used 2,5 µM concentration of working primer solution. It always worked.. So today i made exactly what you've said: i took 10µl from stock (100µM) and combined with 90 µl water. Now the PCR is taking place We'll see what will happen. In my lab everyone dilute primers with water. And i don't think that's great.

Best Wishes!

this is what i get. all the primers are new and diluted as mentioned above.. Seems that there's overloading in the lanes..
Have any idea?
P.S. all the reaction conditions were the same as always - before, using the same conditions i got good results..

PCR mix:
polymerase 0.5 U
buffer 1X
0.25 mM dNTP
primer 0.8 uM
template 50ng

20uL total

Do you have a new (never used) template or did the same one worked before? Did you try to dilute template or use less primer? Did you change anything else besides the primers (and maybe template)?

Trof on Tue Jun 21 11:22:06 2011 said:

thanks for your reply.
no, the templates are the same.. i've tried to dilute primers 2-fold, 3-fold and so on.. now trying to adjust annealing temperatures (besides, the current annealing temperatures are the same i used before).. S*it happens :/