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Help with PCR please !

Help with PCR please ! - PCR - Polymerase Chain Reaction Forum

Help with PCR please ! - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 12-12-2011, 09:12 PM
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Default Help with PCR please !



Hi,

I have a question about making solutions etc

(Stock solution) I have a 794 pmol/ul of my forward primer, which is x100 and I need to make a x10 working solution which i will use for the PCR reaction. I need to have 2.5 ul of the forward primer for the reaction.

So how will I make a x10 working solution from a x100 stock solution ?

and also, what would be the concentration of this primer? how can you calculate concentration of it ?

Please someone help as I am stuck and this is really important for me.
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  #2  
Old 12-13-2011, 01:21 PM
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Default Re: Help with PCR please !

If you have a 100x stock and you want a 10x working solution, just dilute it 10x. Ie. add 9 parts of dH20/any other dilution buffer to 1 part of your stock solution.

Most primers these days comes in standardised stocks, which is 100 uM. Then you dilute it 10x to get 10 uM. Your primer of 794 pmol/u is likely 100 uM, since you said it is a 100x stock (you'll need to do some calculations to figure out the molecular weight of the sequence etc and then the dilution factor).

To calculate your concentration of the primer in a reaction,
primer concentration x (volume used in reaction) / total reaction volume.

E.g. : you used 2.5 ul of a 10 uM primer in a single PCR reaction volume of 50 uL.
10 uM x 2.5 ul / 50 ul = 0.5 uM
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The Following User Says Thank You to jonoave For This Useful Post:
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  #3  
Old 12-13-2011, 06:03 PM
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Default Re: Help with PCR please !

Quote:
Originally Posted by jonoave View Post
If you have a 100x stock and you want a 10x working solution, just dilute it 10x. Ie. add 9 parts of dH20/any other dilution buffer to 1 part of your stock solution.

Most primers these days comes in standardised stocks, which is 100 uM. Then you dilute it 10x to get 10 uM. Your primer of 794 pmol/u is likely 100 uM, since you said it is a 100x stock (you'll need to do some calculations to figure out the molecular weight of the sequence etc and then the dilution factor).

To calculate your concentration of the primer in a reaction,
primer concentration x (volume used in reaction) / total reaction volume.

E.g. : you used 2.5 ul of a 10 uM primer in a single PCR reaction volume of 50 uL.
10 uM x 2.5 ul / 50 ul = 0.5 uM
Thank you ! you explained it very well I understand now
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  #4  
Old 12-14-2011, 06:58 PM
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Default Re: Help with PCR please !

c1xv1=c2xv2
(1) initial concentration
(2) final concentration

mahmoud manama
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