Can anybody please tell me the difference in tris base and tris Cl?
What do you use in Primer suspensions?
Will any body explain with real experiences

Thanks

Hi There,
First Q. Tirs is Trisma base:
Synonyms: 2-Amino-2-(hydroxymethyl)-1,3-propanediol
Tris base
Tris(hydroxymethyl)aminomethane
Trometamol
Molecular Formula: NH2C(CH2OH)3

It has a basic pH when you desolve it in water arround pH11.
Usualy when used in order to get to the desirble pH you need to titer it to gain lower then pH11 you have to use HCl. Then you can get PH of I.e. pH7.5 etc.
There for it is called Tris-HCl

Now please explain to me what are your other Q.
Guy

Thank You Guy

My other Q is
We have bought some oligonucleotides (primers for meat species identification)
We need to resuspend it in TE buffer
For TE buffer what should we add as tris ? tris base or tris Cl ?

After we dissolve them in TE (still hasnt) we get a primers in 100uM (micro molar) dialution
For the PCR, we need 2pmol in 0.15ul (according to literature)

this dilution is complicative
Is there an experience in primer dilution inPCR ? if yes Please explain

Thanks
Dilipfernando

Dilip said:

Thank You Guy My other Q is We have bought some oligonucleotides (primers for meat species identification) We need to resuspend it in TE buffer For TE buffer what should we add as tris ? tris base or tris Cl ? After we dissolve them in TE (still hasnt) we get a primers in 100uM (micro molar) dialution For the PCR, we need 2pmol in 0.15ul (according to literature) this dilution is complicative Is there an experience in primer dilution inPCR ? if yes Please explain Thanks Dilipfernando

If you have to use TE to resuspend your primers then I would recommend that you make up a bottle of 1M Tris-HCl at say pH7.5 or 8.0, as guy described. After autoclaving to sterilize remove a small amount of the 1M tris and check that the pH is still correct. If it's OK then you can make up some TE.

I think people usually use 10mM Tris/1mM EDTA. I use a 0.5M EDTA solution as a stock for this (requires a lot of NaOH pellets to get this concentration of EDTA). One you have made your TE solution of the desired pH, autoclave it, then it's ready to use to resuspend your primers.

Personally, I have switched back to using Millipore filtered sterile water to resuspend primers. This is because if they are used for sequencing then it's better not to have any EDTA in the reaction, and the feared degradation of DNA in dH2O (due to its slightly acidic pH) is not a problem if you keep your stock primers frozen at -20 degrees C. But this is just my opinion so if your SOP says TE then you'd better use TE.

For the calculation:



You have a stock concentration of 100M

You want 2 pmoles in 0.15L
= 13.3 pmoles in 1 L
= 13.3 nmoles in 1 mL
= 13.3 moles in 1 L = 13.3M


Dilution factor: 100/13.3 = 7.519

so use 1L of your stock 100M primer and add 6.519 L TE
(scale up as required)

thanks a lot
I'll take them to consideration
I'll discuss with my supervisor with this information
it seems pretty good and easy calculation

I'll visit you when ever I need you
Thanks again
Dilip fernando

parvoman said:

If you have to use TE to resuspend your primers then I would recommend that you make up a bottle of 1M Tris-HCl at say pH7.5 or 8.0, as guy described. After autoclaving to sterilize remove a small amount of the 1M tris and check that the pH is still correct. If it's OK then you can make up some TE. I think people usually use 10mM Tris/1mM EDTA. I use a 0.5M EDTA solution as a stock for this (requires a lot of NaOH pellets to get this concentration of EDTA). One you have made your TE solution of the desired pH, autoclave it, then it's ready to use to resuspend your primers. Personally, I have switched back to using Millipore filtered sterile water to resuspend primers. This is because if they are used for sequencing then it's better not to have any EDTA in the reaction, and the feared degradation of DNA in dH2O (due to its slightly acidic pH) is not a problem if you keep your stock primers frozen at -20 degrees C. But this is just my opinion so if your SOP says TE then you'd better use TE. For the calculation: You have a stock concentration of 100M You want 2 pmoles in 0.15L = 13.3 pmoles in 1 L = 13.3 nmoles in 1 mL = 13.3 moles in 1 L = 13.3M Dilution factor: 100/13.3 = 7.519 so use 1L of your stock 100M primer and add 6.519 L TE (scale up as required)

Dear parvoman

thank you for your reply

we found your calculation is appealing to our matter

But still I cant understand how the "dilution factor" is derived and used here
will you please explain it a bit more

Thanks