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Diluting Ampicillin into LB Agar

LPS Flow Cytometry

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#1 bioforum

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Posted 21 March 2009 - 07:33 AM

Today was a very bad day at the university... beside that some students answered in the quiz question that if you get blue color in Gram staining that means your bacteria is Gram negative :huh: I noticed most of the students including myself had trouble with the dilution process. So I thought maybe I'd make a quick explanation for what is dilution and how it is done and all the ratios and other confusing stuff. I tried my best to simplify it for people who find trouble in doing their homeworks, labwork, or other calculation problems regarding dilution questions and solutions.

So let's start out with definitions:

Dilution: is the mixing of a small accurately measured sample with a large volume of sterile water or normal saline called (diluents or dilution blank)

Laws:

Dilution = V of Sample / Total V of (sample + diluent)

Dilution Factor = Total V of (sample + diluent) / V of sample
** or we can simply say the reciprocal of Dilution

Starting off with this simple example to understand how these laws are applied.


Posted Image


It has been known that if we use a larger volume we obtain a more accurate dilution.
So for better results, we use 1:1000 dilution. And that is by adding 1ml of sample to 999 ml of diluent. But practically we cannot use 999 ml of diluent. So we do what is called a serial dilution.

Serial Dilution: is a dilution made of a series of smaller dilution, and the total dilution is the product of each dilution in the series.

To understand this more, let's see this example.


Posted Image

Posted Image


Here's an example combining all of the above.


Posted Image


I hope this brief explanation proves helpful to you...let me know if i've made any mistakes... B)

-Property of Yulia-


thank u for your explanation..

what makes me always confused is the last volume used for inoculation (0.1ml in your example)..

-strawberry-



Yeah that's why i almost jumped with 'HAVE' there :blush: sorry about that
i hope my mini-tutorial and examples helped you through it strawberry...:)

-Property of Yulia-



I'm very thankful to you to provide the easier explaination...it's true that many undergrad students have problem in dilution..and the worst thing is if the lecturers/tutors assume that the students are familiar with the concept. it is true that they have learned it in high school, but most of the time they will forget..

Regarding the example 2 given, you dilute a sample (10 ml the final volume) 10x10x10=10 to the power of 3. and u inoculate 0.1 ml in your medium.so supposed the final volume of your medium is 1ml right?

-kent19-

This post has been promoted to an article

#2 BHARGAVI

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Posted 23 March 2009 - 03:09 AM

Dear Sir/Madam,

Please explain me the conversion of % to ppm. I am damn confused.

#3 MaggieRoara

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Posted 24 March 2009 - 01:28 AM

Dear Sir/Madam,

Please explain me the conversion of % to ppm. I am damn confused.


ppm means part per million. there fore x ppm = x parts of something to a million parts of diluent. % is something like "parts per hundred" so if its a 10% SDS solution. Its 10 g per hundres parts diluent which is dH20.

#4 Niraj

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Posted 25 April 2009 - 03:52 PM

Thanks for nice explanation!!!

#5 Jka83

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Posted 21 July 2009 - 02:14 AM

Nice stuff!


Perhaps you can help me with this:


For real time pcr (detection of virus), one wants to measure the sensitivity of the assay by determining the minumun amount of viral copies per reaction that the assay is able to detect.
So, lets say I have 8 ng/ul of target RNA in the sample (measured by nanospec). By using this formula:
Transcript length: 75nucleotides
Concentration: 8 ng/ul = 8 x 10-9 g/ul
Calculation: (8 x 10-9 g/ul / [75x 340]) x 6.022 x 10e23 =about 1,8e11 molecules per ul

I dilute this by factor 10-9 and I get 188 molecules per ul.

My PCR mix is 25ul that includes 2ul of this RNA sample. My assay is able to detect this dilution so can I say that the detection limit is 376 copies PER REACTION (I put 2 microliters and 2X 188/ul=376) or is the limit this 376 divided by the reaction volume of 25??

Thank you for your help!!

#6 lyok

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Posted 21 July 2009 - 03:30 AM

Nice stuff!


Perhaps you can help me with this:


For real time pcr (detection of virus), one wants to measure the sensitivity of the assay by determining the minumun amount of viral copies per reaction that the assay is able to detect.
So, lets say I have 8 ng/ul of target RNA in the sample (measured by nanospec). By using this formula:
Transcript length: 75nucleotides
Concentration: 8 ng/ul = 8 x 10-9 g/ul
Calculation: (8 x 10-9 g/ul / [75x 340]) x 6.022 x 10e23 =about 1,8e11 molecules per ul

I dilute this by factor 10-9 and I get 188 molecules per ul.

My PCR mix is 25ul that includes 2ul of this RNA sample. My assay is able to detect this dilution so can I say that the detection limit is 376 copies PER REACTION (I put 2 microliters and 2X 188/ul=376) or is the limit this 376 divided by the reaction volume of 25??

Thank you for your help!!


Most likely a silly question, but where do you get the 340 , 6.022 and 10e23 from?

Edited by lyok, 21 July 2009 - 03:30 AM.


#7 Dr Teeth

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Posted 21 July 2009 - 11:09 AM

<p>Today was a very bad day at the university... beside that some students answered in the quiz question that if you get blue color in Gram staining that means your bacteria is Gram negative ;) I noticed most of the students including myself had trouble with the dilution process. So I thought maybe I'd make a quick explanation for what is dilution and how it is done and all the ratios and other confusing stuff. I tried my best to simplify it for people who find trouble in doing their homeworks, labwork, or other calculation problems regarding dilution questions and solutions.

So let's start out with definitions:

Dilution: is the mixing of a small accurately measured sample with a large volume of sterile water or normal saline called (diluents or dilution blank)

Laws:

Dilution = V of Sample / Total V of (sample + diluent)

Dilution Factor = Total V of (sample + diluent) / V of sample
** or we can simply say the reciprocal of Dilution

Starting off with this simple example to understand how these laws are applied.


Posted Image


It has been known that if we use a larger volume we obtain a more accurate dilution.
So for better results, we use 1:1000 dilution. And that is by adding 1ml of sample to 999 ml of diluent. But practically we cannot use 999 ml of diluent. So we do what is called a serial dilution.

Serial Dilution: is a dilution made of a series of smaller dilution, and the total dilution is the product of each dilution in the series.

To understand this more, let's see this example.


Posted Image

Posted Image


Here's an example combining all of the above.


Posted Image


I hope this brief explanation proves helpful to you...let me know if i've made any mistakes... :)</p> <div>-Property of Yulia-</div><hr>
<p>thank u for your explanation..


</p> <div>-kent19-</div>




This does contain mistakes. In the description for example 3, the author shows that 43 g of food = 43 ml. This is only true if the density of the food = 1g/ml. Since we are not given the density of the food nor told that it is equal to that of pure water at 4C, the calculation cannot be performed as described without undue assumptions by the author.

Edited by Dr Teeth, 21 July 2009 - 11:10 AM.


Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#8 Jka83

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Posted 22 July 2009 - 02:32 AM

Nice stuff!


Perhaps you can help me with this:


For real time pcr (detection of virus), one wants to measure the sensitivity of the assay by determining the minumun amount of viral copies per reaction that the assay is able to detect.
So, lets say I have 8 ng/ul of target RNA in the sample (measured by nanospec). By using this formula:
Transcript length: 75nucleotides
Concentration: 8 ng/ul = 8 x 10-9 g/ul
Calculation: (8 x 10-9 g/ul / [75x 340]) x 6.022 x 10e23 =about 1,8e11 molecules per ul

I dilute this by factor 10-9 and I get 188 molecules per ul.

My PCR mix is 25ul that includes 2ul of this RNA sample. My assay is able to detect this dilution so can I say that the detection limit is 376 copies PER REACTION (I put 2 microliters and 2X 188/ul=376) or is the limit this 376 divided by the reaction volume of 25??

Thank you for your help!!


Most likely a silly question, but where do you get the 340 , 6.022 and 10e23 from?


There are no silly questions.
The formula can be found from here:
http://www1.qiagen.c...uantifying.aspx
"RNA standards"

#9 lyok

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Posted 22 July 2009 - 03:50 AM

Ah I see, thanks for the link.

I do have a question: what do you mean with: I dilute this by factor 10-9 and I get 188 molecules per ul.

My PCR mix is 25ul that includes 2ul of this RNA sample. My assay is able to detect this dilution so can I say that the detection limit is 376 copies PER REACTION (I put 2 microliters and 2X 188/ul=376) or is the limit this 376 divided by the reaction volume of 25??

I do not understand your question.


In the end you simply have 376 molecules per 25l and and you can detect this.

You can then say , I think , that you will detect 376 molecules per 25l (=per reaction) or 376 molecules/25l = X molecules per l.

Is this not the same?

#10 Maddie

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Posted 17 November 2009 - 04:02 PM

And for the lazy ones, like me ;)

http://www.graphpad....olarityform.cfm
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#11 aimikins

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Posted 20 November 2009 - 03:49 PM

I have a pile of bookmarked calculations tools, although I think it's VERY important to learn how to do these so that you understand the principle and are therefore less likely to make errors. I also have a pile of other useful sites in my 'toolbox' bookmarks, here:

http://www.promega.c...ath/default.htm
http://www.cellbiol....t_sequence.html
http://www.biocarta....genes/index.asp
http://www.hprd.org/
http://www.graphpad....olarityform.cfm
http://www.fauvet.fa...uts/mouseHO.htm
http://www.sigmaaldr...al-buffers.html
http://info.med.yale...d/tavi/PCR.html
http://www.thelabrat.../reagents.shtml
http://molbiol.ru/en...ipts/01_05.html

and, holy moly, whatever you do, don't forget that there's a lot of really good information to be found here: www.protocol-online.org in the main site, not just the forum :wacko:
"it is a miracle that curiosity survives formal education" -A.E.

#12 ufumes

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Posted 18 February 2010 - 03:43 PM

I have a pile of bookmarked calculations tools, although I think it's VERY important to learn how to do these so that you understand the principle and are therefore less likely to make errors. I also have a pile of other useful sites in my 'toolbox' bookmarks, here:

http://www.promega.c...ath/default.htm
http://www.cellbiol....t_sequence.html
http://www.biocarta....genes/index.asp
http://www.hprd.org/
http://www.graphpad....olarityform.cfm
http://www.fauvet.fa...uts/mouseHO.htm
http://www.sigmaaldr...al-buffers.html
http://info.med.yale...d/tavi/PCR.html
http://www.thelabrat.../reagents.shtml
http://molbiol.ru/en...ipts/01_05.html

and, holy moly, whatever you do, don't forget that there's a lot of really good information to be found here: www.protocol-online.org in the main site, not just the forum :wacko:


Your site suggestions are quite helpul, but it appears that you are missing out http://nigerianbioscientist.com- It contains a properly categorized listings of free online bioresources- Online tutorials, science databases, online libraries, and lots more. You can check it out right away

#13 shyarasu

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Posted 21 February 2010 - 08:41 PM

<p>Today was a very bad day at the university... beside that some students answered in the quiz question that if you get blue color in Gram staining that means your bacteria is Gram negative :D I noticed most of the students including myself had trouble with the dilution process. So I thought maybe I'd make a quick explanation for what is dilution and how it is done and all the ratios and other confusing stuff. I tried my best to simplify it for people who find trouble in doing their homeworks, labwork, or other calculation problems regarding dilution questions and solutions.

So let's start out with definitions:

Dilution: is the mixing of a small accurately measured sample with a large volume of sterile water or normal saline called (diluents or dilution blank)

Laws:

Dilution = V of Sample / Total V of (sample + diluent)

Dilution Factor = Total V of (sample + diluent) / V of sample
** or we can simply say the reciprocal of Dilution



Hi, Does anybody use lipopeptide antigens in immunological reactions? What tests are best suited for them? Can we use MQ water to reconstitute synthetically prepared lipopetides? What about culture medium?

Starting off with this simple example to understand how these laws are applied.


Posted Image


It has been known that if we use a larger volume we obtain a more accurate dilution.
So for better results, we use 1:1000 dilution. And that is by adding 1ml of sample to 999 ml of diluent. But practically we cannot use 999 ml of diluent. So we do what is called a serial dilution.

Serial Dilution: is a dilution made of a series of smaller dilution, and the total dilution is the product of each dilution in the series.

To understand this more, let's see this example.


Posted Image

Posted Image


Here's an example combining all of the above.


Posted Image


I hope this brief explanation proves helpful to you...let me know if i've made any mistakes... :)</p> <div>-Property of Yulia-</div><hr>
<p>thank u for your explanation..

what makes me always confused is the last volume used for inoculation (0.1ml in your example)..</p> <div>-strawberry-</div><hr>

<p>Yeah that's why i almost jumped with 'HAVE' there :blush: sorry about that
i hope my mini-tutorial and examples helped you through it strawberry...:)</p> <div>-Property of Yulia-</div><hr>

<p>I'm very thankful to you to provide the easier explaination...it's true that many undergrad students have problem in dilution..and the worst thing is if the lecturers/tutors assume that the students are familiar with the concept. it is true that they have learned it in high school, but most of the time they will forget..

Regarding the example 2 given, you dilute a sample (10 ml the final volume) 10x10x10=10 to the power of 3. and u inoculate 0.1 ml in your medium.so supposed the final volume of your medium is 1ml right?
</p> <div>-kent19-</div>



#14 ikwana

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Posted 18 August 2010 - 10:16 PM

i'm now trying to find how to determine the standard concentration (dially disulphide, diallyl sulphide, ajoene) for Gas chromatography analysis.

and.. sumbody told me to find it, u must calculate from Rf factor..

then i go..." what da heck is Rf? how to calculate it??" :o

i try to find from the internet.. but.. nothin.. <_<

my fren said.. rf is sumthing u must calculate from the graph... then i go..."wut? :o graph? :huh: ?"

GC... for chemistry student... not me.. i havent learn it.. i've only use it twice.. :(

please guys.. help me..... how? how? how?

#15 FLC

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Posted 02 November 2010 - 07:00 AM

Hello!

I have done serial dilutions of my Ab for IHC, but the staining doesn't make sense. Some lower concentrations have stronger signal the the higher ones. Can you check if I made the right dilutions?
I started with a 1:1000 solution and I need final volumes of 320 uL to each dilution.

For a 1:2000, I took 160 uL from the 1:1000 and add 160 uL of diluting solution.

For a 1:2500, I took 128 uL from the 1:1000 and 192 uL of diluting solution.

And for a 1:3000, I took 107 uL from 1:1000 and 213 uL of diluting sol.

My calculus are wrong?
Thank you very much for your help




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