confirmation for primers dilution - (Oct/15/2014 )

Hi everybody,

 

My question is trivial but I am carrying out primers reconstitution and just want to be sure to avoid mistakes.

 

Exemple for one primer:

 

according to my primer synthesis repport, I sould add 129µl of water to obtain a 100µM solution (stock tube).

Then I should prepare 100 µl of a 1:10 solution =10µM=10picomol/µl....right?

I have to take 10 µl of my stock solution and add 90ml to obtain a 10µM solution (second tube)

 

To perform my PCR, I need my primer at 6µM, then I did calculation as in first time

I need 60µl from the second tube and add 40µl of water to obtain my working tube at 6µM and then ready to go

 

 

Is it right or there is any mistake there?

 

thank's in advance 

There are a couple of mistakes
 

Wan2Know on Wed Oct 15 10:22:42 2014 said:

Hi everybody,
 
My question is trivial but I am carrying out primers reconstitution and just want to be sure to avoid mistakes.
 
Exemple for one primer:
 
according to my primer synthesis repport, I sould add 129µl of water to obtain a 100µM solution (stock tube).
Then I should prepare 100 µl of a 1:10 solution =10µM=10picomol/µl....right?

correct.
 

Wan2Know on Wed Oct 15 10:22:42 2014 said:

I have to take 10 µl of my stock solution and add 90ml to obtain a 10µM solution (second tube)

No, 10 ul + 90 ul (not ml), this is a 1:10 dilution as you said above, not a 1:11000

Wan2Know on Wed Oct 15 10:22:42 2014 said:

To perform my PCR, I need my primer at 6µM, then I did calculation as in first time
I need 60µl from the second tube and add 40µl of water to obtain my working tube at 6µM and then ready to go
 
 
Is it right or there is any mistake there?
 
thank's in advance

6 uM is a very high concentration for a PCR, usually primers are used between 0.1 and 0.6 uM, and this concentration should be obtained by adding working stocks of primers to the reaction mix. If you generate 6 uM as you have above and then dilute this by adding it to a reaction mix, it will no longer be 6 uM!

Hi bob1,

Thank's a lot for your response and correction, it helps a lot.

First mistake is a big confusion, the proof that you are very attentive :-).

 

The second one is disturbing me, because primers at 6µM is indicated in my protocol as well as in an article dealing with my experiment. Authors repport: The PCR amplifications (multiplex) were performed using a mixture of all four primers at 6µM each (Zhan et al., 2002).

May be is usefull to give you reaction mix volumes :

 

dNTP (600µM)..5µl

Buffer X10.........5µl

Taq....................0,25µl

Primer 1F (6µM)...2,5µl

Primer 1R (6µM)...2,5µl

Primer 2F (6µM)...2,5µl

Primer 2R (6µM)...2,5µl

MQ water..............28,75µl

DNA (1ng/µl).........1µl

 

Total.......................50µl

 

What do you think about? looking for your advice...thank's a lot

Hi again,

I did calculation for the final primers concentration , I don't know if it's correct but primers should be at 0,3µM in 50µl PCR mix

 

???

The people who wrote your PCR protocol (for whatever reason) decided to dilute their primer stock to 6 uM. This is an unusual stock concentration, and I would not choose it. Far more standard is to choose 10 uM stock. In any case, you can adjust for this in your PCR reaction setup by reducing the amount of stock primer used. I would do this:

 

dNTP (600µM)..5µl

Buffer X10.........5µl

Taq....................0,25µl

Primer 1F (10µM)...2,1µl

Primer 1R (10µM)...2,1µl

Primer 2F (10µM)...2,1µl

Primer 2R (10µM)...2,1µl

MQ water..............30,35µl

DNA (1ng/µl).........1µl

 

Total.......................50µl

Hi Veteran, 

 

Thank's for your reply. As it's the first time I perform this kind of protocol and according to your advice, I will try 2 mixes with 6µM and 10µM (dilution at 1:10 right?) and will inform you about the issue, this is to avoid any doubt if 6µM will not work

 

Thank's again for your proposition

 

Regards 

As Phage pointed out - the people who wrote your protocol used a 6 uM stock (not final concentration). The final concentration is 0.3 uM... if the reactions work using the 6 uM stock, they should also work by adding 10 uM stock to the give the same final concentration.

Hi Bob, thank's to be here again.
Actually I've already performed PCR with previous conditions and it works, but I was in a host lab and all reagents were ready to use and more over I was assisted by an experimented person. Now I have to repeat it again for remaining DNA samples and should do it alone. That's why I am doubting and not confident

Hi again Bob and phage, I finally performed my PCR's with bands revelation. I am not used with molecular techniques yet and lot of things are new to me.
this is my gel and it's not good, I used 1kb step ladder ( Promega) and bands does not appear as expected. Do you have an idea about the problem?

A picture would be useful. What is the problem with the bands exactly? Size? appearance of the bands in the gel? multiple banding? etc.