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A problem with basic concentrations and serial dilution calculations


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#1 Vg12th

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Posted 06 January 2014 - 08:33 AM

Dear community,

 

I've recently joined a lab for an internship and find myself lost with basic math problems! I'm feeling quite embarassed to ask this but I need to get these basics in my pocket.

1. For PCR I need 0.3uM concentration of primers. We keep our stock at 10uM. How much do I pipet?

2. Serial dilutions when the amount is too small to pipet eg. 0.056ul.

 

 



#2 bob1

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Posted 06 January 2014 - 11:49 AM

OK - this is more homework for you, so I'm not giving you the answers fully - you'll have to do some thinking for yourself.

 

How big a volume would you expect to have for a PCR (for 1 tube)? - so what sort of volume would you be pipetting for the primers (ml? l? ul? nl?)? 

 

uM is micro mol per litre, how many pmol of primers will be in each reaction?  And what volume would that work out to be?

 

Serial dilutions - you could do 1:10 dilution or any other multiple to get it to a pipettable volume (0.5 ul and above).  1:10 and 1:2 are the easiest as they are kind of intuitive (multiply by 10 or 2 respectively)

 

For dilutions the best equation to use is Vi x Ci = Vf x Cf where V and C are volume and concentration and i and f are initial and final respectively.



#3 Vg12th

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Posted 06 January 2014 - 02:03 PM

 

OK - this is more homework for you, so I'm not giving you the answers fully - you'll have to do some thinking for yourself.

 

How big a volume would you expect to have for a PCR (for 1 tube)? - so what sort of volume would you be pipetting for the primers (ml? l? ul? nl?)? 

 

uM is micro mol per litre, how many pmol of primers will be in each reaction?  And what volume would that work out to be?

 

Serial dilutions - you could do 1:10 dilution or any other multiple to get it to a pipettable volume (0.5 ul and above).  1:10 and 1:2 are the easiest as they are kind of intuitive (multiply by 10 or 2 respectively)

 

For dilutions the best equation to use is Vi x Ci = Vf x Cf where V and C are volume and concentration and i and f are initial and final respectively.

The volume for PCR will be in the microlitres. We'll be using 25ul total reaction. 

0.3uM = 0.3pico mol / ul. Does this mean that from my stock of 10uM, pipetting 1ul will give me 0.3pico mol for my reaction tube? 

I normally do use civ1 =c2v2 in calculations but here there is no given vf (as I'm designing the experiment and I just know it should be below 2ul)

 

For the serial dilution. If I multiply by 10 I can reach a pipettable amount. So from 0.056/1, multiplied by 10 I get 0.56/10 dilution.I can pipet this by adding 0.56ul in 10(-0.56)ul. From this do I add 1ul to my final tube? 

 

Sorry if it's all over the place, I am a confused about this topic.



#4 bob1

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Posted 06 January 2014 - 05:31 PM


The volume for PCR will be in the microlitres. We'll be using 25ul total reaction. 

0.3uM = 0.3pico mol / ul. Does this mean that from my stock of 10uM, pipetting 1ul will give me 0.3pico mol for my reaction tube? 

I normally do use civ1 =c2v2 in calculations but here there is no given vf (as I'm designing the experiment and I just know it should be below 2ul)

 

For the serial dilution. If I multiply by 10 I can reach a pipettable amount. So from 0.056/1, multiplied by 10 I get 0.56/10 dilution.I can pipet this by adding 0.56ul in 10(-0.56)ul. From this do I add 1ul to my final tube? 

 

Sorry if it's all over the place, I am a confused about this topic.

 

Correct - 1 ul will give you 0.3 pmol - this should enable you to get the right volume to use.  If you don't have a Vf, but you do know the amount you want in total, use V=n/C  where n is the amount you want.

 

For a dilution 1:10 means take 1 volume of sample and make it up to 10 volumes in diluent (e.g. 1 ul sample + 9 ul diluent = 10 ul total) or any multiples thereof.  You would then take 0.56 ul of this (0.056 x 10)...



#5 mdfenko

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Posted 07 January 2014 - 06:03 AM

1ul of 10uM will give 10pmol


Edited by mdfenko, 07 January 2014 - 06:03 AM.

talent does what it can
genius does what it must
i do what i get paid to do

#6 bob1

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Posted 07 January 2014 - 12:04 PM

Oh, yeah, sorry just read the first bit about 0.3 uM...  As Mdfenko says...






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