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Realtime PCR

Realtime PCR - DNA Forum

Realtime PCR - Discuss DNA, the molecule of hereditary. Topics include DNA structure, DNA replication, DNA function.

#1
05-23-2010, 01:47 PM
Pipette Filler

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Realtime PCR

I am really new to qpcr and I need to set up this pcr for 2 different DNA fragments with concentration of 0.1nM. Total volume of each fragment is 5ul.Was told to make a diltution series between 10pM to 0.1nM. I am really confused as to what to do. can someone please help or advice?
#2
05-24-2010, 07:16 PM
Pipette Filler

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Re: Realtime PCR

Essentially you need to amplify the DNA at different concentrations.

you use 5µl of the end solution I'm guessing.
You would seeminglu need to make serial dilutions.

Nanomolar nM 10^-9 molar 0.000,000,00X
Picomolar pM 10^-12 molar 0.000,000,000,00x (commas to help counting dont actually put them in!)

10 pM =0.000,000,000,010 M
0.1 nM=0.000,000,000,1 M

essentially convert the nanoMoles into picoMoles

0.1nM = 100 pM
10pM = 10pM ;-)

So you need a dilution series of 10 and 100pM. I would need to know what you started with to give exact examples but if you had 100pM/0.1nM solution and you need to make 10pM solution you'd do a 1 in ten. Get 45 units of buffer and add 5 units of your 100pM solution. This would dilute it by a factor of 10. (5+45=50, 5 is 10% of 50)

The units depend on what you're working with... ml, µl or whatever.

Hope that helps.
 The Following User Says Thank You to Phototoxin For This Useful Post: rangermat (06-01-2010)
#3
06-01-2010, 11:51 AM
Pipette Filler

Join Date: May 2010
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Re: Realtime PCR

Thanks for your input...was able to figure it out later! Effort really appreciated!

 Tags pcr , realtime

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