Calculation Madness - It is driving me crazy!!! (Jan/08/2010 )
Hi everyone,
I have been working in a lab for about 8 months and I am still really bad at calculations and seriously need some guidance! It has been driving me crazy for a day so I would appreciate any help!! So here I go...
I have a stock of 100ng/uL of DNA, and the DNA is about 100bp, so the molecular weight of it would be 66000Da, which can also be written as... 66000g/mole. I am supposed to add 10nM of the DNA into 220uL of quenching buffer. This quenching buffer will eventually be used to break of emulsions in my selection work.
I am unsure as to whether I am right about this. I am going to be very detailed in my working:
1M = 1 mole/L
10nM = 10 X 10^-9 mole/L
I need to times the concentration with the molecular weight: 10 X 10^-9 mole/L X 66000 g/mole = 6.6 X 10-4 g/L, which is also 0.66 ng/uL
If.... V1C1 = V2C2
0.66 ng/uL X 220uL = V2 X 100ng/uL
V2 = 1.452uL rounded off to 1.5uL
So this means I need 1.5uL of 100ng/uL DNA stock to get 10nM in the 220uL quenching buffer.
Am I right? Please help me to check my working as I tend to make a lot of mistakes in it. I asked a few people and they used different methods to solve this for my experiment and as a result, they all had different answers and I am so confused!
Thanks in advance!!
By the way.. I found this website http://forum.onlineconversion.com/showthread.php?t=11182 and tried to backcalculate but could make no sense out of it..
Calculating it in a slightly different way I got the same number so.... you must be right 
jiajia1987 on Jan 8 2010, 01:24 PM said:
I have been working in a lab for about 8 months and I am still really bad at calculations and seriously need some guidance! It has been driving me crazy for a day so I would appreciate any help!! So here I go...
I have a stock of 100ng/uL of DNA, and the DNA is about 100bp, so the molecular weight of it would be 66000Da, which can also be written as... 66000g/mole. I am supposed to add 10nM of the DNA into 220uL of quenching buffer. This quenching buffer will eventually be used to break of emulsions in my selection work.
I am unsure as to whether I am right about this. I am going to be very detailed in my working:
1M = 1 mole/L
10nM = 10 X 10^-9 mole/L
I need to times the concentration with the molecular weight: 10 X 10^-9 mole/L X 66000 g/mole = 6.6 X 10-4 g/L, which is also 0.66 ng/uL
If.... V1C1 = V2C2
0.66 ng/uL X 220uL = V2 X 100ng/uL
V2 = 1.452uL rounded off to 1.5uL
So this means I need 1.5uL of 100ng/uL DNA stock to get 10nM in the 220uL quenching buffer.
Am I right? Please help me to check my working as I tend to make a lot of mistakes in it. I asked a few people and they used different methods to solve this for my experiment and as a result, they all had different answers and I am so confused!
Thanks in advance!!
almost a doctor on Jan 8 2010, 10:01 PM said:
jiajia1987 on Jan 8 2010, 01:24 PM said:
I have been working in a lab for about 8 months and I am still really bad at calculations and seriously need some guidance! It has been driving me crazy for a day so I would appreciate any help!! So here I go...
I have a stock of 100ng/uL of DNA, and the DNA is about 100bp, so the molecular weight of it would be 66000Da, which can also be written as... 66000g/mole. I am supposed to add 10nM of the DNA into 220uL of quenching buffer. This quenching buffer will eventually be used to break of emulsions in my selection work.
I am unsure as to whether I am right about this. I am going to be very detailed in my working:
1M = 1 mole/L
10nM = 10 X 10^-9 mole/L
I need to times the concentration with the molecular weight: 10 X 10^-9 mole/L X 66000 g/mole = 6.6 X 10-4 g/L, which is also 0.66 ng/uL
If.... V1C1 = V2C2
0.66 ng/uL X 220uL = V2 X 100ng/uL
V2 = 1.452uL rounded off to 1.5uL
So this means I need 1.5uL of 100ng/uL DNA stock to get 10nM in the 220uL quenching buffer.
Am I right? Please help me to check my working as I tend to make a lot of mistakes in it. I asked a few people and they used different methods to solve this for my experiment and as a result, they all had different answers and I am so confused!
Thanks in advance!!
Hey that's cool! Thanks a lot!!
But I would like to know what method you used, mind teaching me?
I did the same, just slightly different.
You need 10nM in 220ul, so from M= moles/V(L) you know you need 2.2x10^-12 moles.
now 2.2x10^-12 moles x 660000 Da = need 1.452x10^-7 grs
you have 100ng in each ul so: 1.452x10-7 / 100x10-9 = 1.452 ul
same thing really 
another way will be.
100ng/ul = 1.51515x10^-6M now apply V1C1=V2C2
so V1 = 10x10^-9 x 220ul / 1.51515x10^-6 = 1.452ul
All the same, as long as you know your units and keep things consistent, you'll get to the same numbers
Great!! Now I know of a few ways to solve it. I am grateful for the guidance!! Thanks so much!!
almost a doctor on Jan 8 2010, 10:51 PM said:
You need 10nM in 220ul, so from M= moles/V(L) you know you need 2.2x10^-12 moles.
now 2.2x10^-12 moles x 660000 Da = need 1.452x10^-7 grs
you have 100ng in each ul so: 1.452x10-7 / 100x10-9 = 1.452 ul
same thing really
another way will be.
100ng/ul = 1.51515x10^-6M now apply V1C1=V2C2
so V1 = 10x10^-9 x 220ul / 1.51515x10^-6 = 1.452ul
All the same, as long as you know your units and keep things consistent, you'll get to the same numbers
This is correct as long as "I am supposed to add 10nM of the DNA into 220uL of quenching buffer" is correct. In other words, if you're supposed to wind up with 10 nM of DNA in a final volume of 221.5 ul.
With such a small amount added, it's not likely to make a difference in this case (the marigin of error in measuring 220 ul is likely to add as much error), but with larger volumes (or differently worded protocols) you'd need to take the additon into account if the final volume was specified. In other words, if your protocol called for you to combine 10 nm of DNA and the quenching buffer in a final volume of 220 ul, you'd need to add your 1.5 ul to 218.5 ul of the buffer.
HomeBrew on Jan 10 2010, 01:13 AM said:
With such a small amount added, it's not likely to make a difference in this case (the marigin of error in measuring 220 ul is likely to add as much error), but with larger volumes (or differently worded protocols) you'd need to take the additon into account if the final volume was specified. In other words, if your protocol called for you to combine 10 nm of DNA and the quenching buffer in a final volume of 220 ul, you'd need to add your 1.5 ul to 218.5 ul of the buffer.
Yes Sir! I get it now!
HomeBrew on Jan 9 2010, 05:13 PM said:
With such a small amount added, it's not likely to make a difference in this case (the marigin of error in measuring 220 ul is likely to add as much error), but with larger volumes (or differently worded protocols) you'd need to take the additon into account if the final volume was specified. In other words, if your protocol called for you to combine 10 nm of DNA and the quenching buffer in a final volume of 220 ul, you'd need to add your 1.5 ul to 218.5 ul of the buffer.
Indeed!! I was always considering 220ul final volume, so you have to add 220 - 1.5 = Volume of buffer (218.5ul).
In the case that you HAVE to add 10nM to 220ul of buffer you can still use C1V1=C2V2 to calculate the volume of DNA you have to add (V1) but in this case your
V2 = volumen of buffer (220ul) + volume of DNA needed (V1)
C1V1=C2(220+V1) V1= 220*C2 / (C1-C2)
almost a doctor on Jan 11 2010, 05:41 PM said:
HomeBrew on Jan 9 2010, 05:13 PM said:
With such a small amount added, it's not likely to make a difference in this case (the marigin of error in measuring 220 ul is likely to add as much error), but with larger volumes (or differently worded protocols) you'd need to take the additon into account if the final volume was specified. In other words, if your protocol called for you to combine 10 nm of DNA and the quenching buffer in a final volume of 220 ul, you'd need to add your 1.5 ul to 218.5 ul of the buffer.
Indeed!! I was always considering 220ul final volume, so you have to add 220 - 1.5 = Volume of buffer (218.5ul).
In the case that you HAVE to add 10nM to 220ul of buffer you can still use C1V1=C2V2 to calculate the volume of DNA you have to add (V1) but in this case your
V2 = volumen of buffer (220ul) + volume of DNA needed (V1)
C1V1=C2(220+V1) V1= 220*C2 / (C1-C2)
Okay great! Now, I know why sentence construction matters so much in science. My lecturer was emphasizing on how we should bring across our point once as it could mean different things!