6 replies to this topic

[nbhu]

Hi guys,
I think i did something silly!
Basically i want to obtain a primer stock of 1ug/ul.
Instead of just adding the same volume as the ug amount, i blindly made up a 100pmol/ul stock by adding the volume the primer company recommended.
E.g.
Yield: (ug): 213
Vol for 100pmol/ul: 317ul
MW (g/mol): 6731

So i added the 317ul and have a 100pmol/ul .... how do i now go about diluting this to obtain a 1ug/ul stock?

Any help would be appreciated!!!

[tea-test]

without calculating it, but just from your description i would say it is not possible anymore.

you have 213µg of primer. so, to get a 1µg/µl dilution you would have to add 213µl, do you agree? but you have already added 317µl....

[Wolverena]

nbhu, on May 1 2009, 12:34 PM, said:

Hi guys,
I think i did something silly!
Basically i want to obtain a primer stock of 1ug/ul.
Instead of just adding the same volume as the ug amount, i blindly made up a 100pmol/ul stock by adding the volume the primer company recommended.
E.g.
Yield: (ug): 213
Vol for 100pmol/ul: 317ul
MW (g/mol): 6731

So i added the 317ul and have a 100pmol/ul .... how do i now go about diluting this to obtain a 1ug/ul stock?

Any help would be appreciated!!!

I think you have about 0.67ug/ul. Can you compensate by adding more ul for that primer when you do your PCR?

[wuxx0153]

tea-test, on May 1 2009, 09:52 AM, said:

without calculating it, but just from your description i would say it is not possible anymore.

you have 213µg of primer. so, to get a 1µg/µl dilution you would have to add 213µl, do you agree? but you have already added 317µl....

As tea-test has said, no way you can back to 1 µg/µl.
However, your primer stock is now 100 µM and as I understand, PCR primer final concentration (in most of my PCR reaction, 0.2 µM) is the one important for the reaction.
You dilute your stock into working solution (in my case 5 µM) and then add diluted primer to PCR master mix to reach final concentration.

Your primer concentration now is better than 1 µg/µl.
Why you have to get 1 µg/µl?

[nbhu]

wuxx0153, on May 1 2009, 07:13 PM, said:

tea-test, on May 1 2009, 09:52 AM, said:

without calculating it, but just from your description i would say it is not possible anymore.

you have 213µg of primer. so, to get a 1µg/µl dilution you would have to add 213µl, do you agree? but you have already added 317µl....

As tea-test has said, no way you can back to 1 µg/µl.
However, your primer stock is now 100 µM and as I understand, PCR primer final concentration (in most of my PCR reaction, 0.2 µM) is the one important for the reaction.
You dilute your stock into working solution (in my case 5 µM) and then add diluted primer to PCR master mix to reach final concentration.

Your primer concentration now is better than 1 µg/µl.
Why you have to get 1 µg/µl?

Well basically it is for a long range pcr, and it specifies 1ug/ul... i guess it will be easy enough compensating for it.
I would add 2ul to the reaction if it was a 1ug/ul stock, so how much do you think i should be adding now?

[nbhu]

nbhu, on May 1 2009, 08:28 PM, said:

wuxx0153, on May 1 2009, 07:13 PM, said:

tea-test, on May 1 2009, 09:52 AM, said:

without calculating it, but just from your description i would say it is not possible anymore.

you have 213µg of primer. so, to get a 1µg/µl dilution you would have to add 213µl, do you agree? but you have already added 317µl....

As tea-test has said, no way you can back to 1 µg/µl.
However, your primer stock is now 100 µM and as I understand, PCR primer final concentration (in most of my PCR reaction, 0.2 µM) is the one important for the reaction.
You dilute your stock into working solution (in my case 5 µM) and then add diluted primer to PCR master mix to reach final concentration.

Your primer concentration now is better than 1 µg/µl.
Why you have to get 1 µg/µl?

Well basically it is for a long range pcr, and it specifies 1ug/ul... i guess it will be easy enough compensating for it.
I would add 2ul to the reaction if it was a 1ug/ul stock, so how much do you think i should be adding now?

actually, sorry, i got that wrong... from the 1ug/ul, i dilute this to 60ng/ul and use 2ul....
its harder doing this when you are following someone elses protocols and the math just makes my head go all fuzzy :s

[almost a doctor]

nbhu, on May 1 2009, 08:30 PM, said:

nbhu, on May 1 2009, 08:28 PM, said:

wuxx0153, on May 1 2009, 07:13 PM, said:

tea-test, on May 1 2009, 09:52 AM, said:

without calculating it, but just from your description i would say it is not possible anymore.

you have 213µg of primer. so, to get a 1µg/µl dilution you would have to add 213µl, do you agree? but you have already added 317µl....

As tea-test has said, no way you can back to 1 µg/µl.
However, your primer stock is now 100 µM and as I understand, PCR primer final concentration (in most of my PCR reaction, 0.2 µM) is the one important for the reaction.
You dilute your stock into working solution (in my case 5 µM) and then add diluted primer to PCR master mix to reach final concentration.

Your primer concentration now is better than 1 µg/µl.
Why you have to get 1 µg/µl?

Well basically it is for a long range pcr, and it specifies 1ug/ul... i guess it will be easy enough compensating for it.
I would add 2ul to the reaction if it was a 1ug/ul stock, so how much do you think i should be adding now?

actually, sorry, i got that wrong... from the 1ug/ul, i dilute this to 60ng/ul and use 2ul....
its harder doing this when you are following someone elses protocols and the math just makes my head go all fuzzy :s

Hi, by adding 317µl to your primer this is now at 0.672µg/µl, so is 10x more concentrated than you need it (60ng/µl).  You need to make a ~1:10 dilution of your stock to have the primer at 60ng/µl (actually 1:10.3).  This is, 8.9µl of you stock into a final 100µl volume.  Then you can use 2µl for your PCR reaction.