16 replies to this topic

[DNRDNR]

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

[GNANA]

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..

Edited by GNANA, 17 June 2011 - 07:47 AM.

[DNRDNR]

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

Edited by DNRDNR, 17 June 2011 - 10:12 AM.

[GNANA]

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.

Edited by GNANA, 17 June 2011 - 12:23 PM.

[DNRDNR]

GNANA, on 17 June 2011 - 12:09 PM, said:

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)? if so ok,, better have a check over it and proceed...

hope this helps.

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

[Trof]

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?

[DNRDNR]

Trof, on 18 June 2011 - 01:27 AM, said:

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?

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!

[DNRDNR]

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

Attached Thumbnails

• 

[Trof]

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)?

[DNRDNR]

Trof, on 21 June 2011 - 03:22 AM, said:

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)?

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 :/

[almost a doctor]

I think your gel is well overloaded.  How much of your PCR product are you running?  Maybe you could try with less... I've seen this happen with RNA, sometimes what looks like a degraded sample (smear) is actually the result of an overload, easily fixed by running a new gel with lower sample volume.

just my 2 cents  

[Ameya P]

DNR,

Other than overloading of samples, I think there is too much template DNA to start with.

• You could try a 5 fold, 10-fold or 100 fold dilution of the template you use for the PCR.
  
• Also, if you total volume is 20 ul, you could also consider lowering the Taq you use to 0.2- 0.3 U.
  
• Because you mentioned, ISSR, I am presuming you are working with plant DNA and if so, you could consider treating your template with RNAse, before proceeding to PCR. It will get rid of smears that you are seeing on the lower end of the gel.
  
  Does DNR, have anything to do with DNR Israel ?

[Evanescence]

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
[/quote]


they dont give you the Tm? i guess u need to change the annealing temperature 1-2C higher than what u was used now...smearing mean the the pcr amplifying but unspecific bind...10uM primer concentration is good working conc...

[DNRDNR]

Thank for your all replies!

Seems, the problem is solved. Yes, they give me Tm. It says 55 C (on data sheet). Don't trust it!
Tried different temepratures with thermogradient PCR. And wow The annealing step should have done at 56C.

Besides, i received the same primer some time ago - and i was given instructions to set annealing temperature to 50C. It worked! But then, when i bought THE SAME primer, the things went wrong.

In general, yes, it was the annealing temperature.

Thanks for your contribution

[Trof]

Yes, Metabion calculates the Tm with a different method than primer3 which I use. I don't look at it either, Ta -4 degs from primer3 Tm works for me.