8 replies to this topic

[starvingstudent]

I have a question regarding measuring concentration on the nanodrop. Does it matter how much volume (more than 1 microliter) I load onto the machine?  If I load more than 1 microliter onto the nanodrop,say 2 microliters of a DNA sample and the resulting concentration is 140 ng/microliter, does that mean that there are 70 ng in each microliter of my sample or is the concentration the machine gives me (140 ng/microliter) the actual concentration of my sample regardless of how much I load?
Thanks in advance for any clarification you all can provide!

[Vini]

starvingstudent, on Jul 22 2009, 10:16 AM, said:

I have a question regarding measuring concentration on the nanodrop. Does it matter how much volume (more than 1 microliter) I load onto the machine?  If I load more than 1 microliter onto the nanodrop,say 2 microliters of a DNA sample and the resulting concentration is 140 ng/microliter, does that mean that there are 70 ng in each microliter of my sample or is the concentration the machine gives me (140 ng/microliter) the actual concentration of my sample regardless of how much I load?
Thanks in advance for any clarification you all can provide!

No, it does not matter whether u load 1 or 2 ul. Thats because u get the concentration in ng/ul. therefore, volume is automatically taken care of. to remove pipetting error, i prefer taking 2 ul of my sample.

[jiajia1987]

THe concentration that nanodrop gives is in per ul, so it doesnt matter how much you put in. I use 2uL too.

[swanny]

Ditto, 2 ul. 1 ul is a bit fiddly.

[fishdoc]

Agreed. 2 ul for the sample and 2 ul for the water blank. Only use 1.5 ul to initialize the machine.

[jiajia1987]

fishdoc, on Jul 23 2009, 09:52 AM, said:

Agreed. 2 ul for the sample and 2 ul for the water blank. Only use 1.5 ul to initialize the machine.

Hi fishdoc,

What I always do is to add 2ul and den close the cover and initialize the machine. After that is done, I just click on blank. This is alright, right? That was what my supervisor told me. Didnt know that I had to take a small volume to initialize first, den clean the machine, and add again to blank.

[fishdoc]

jiajia1987, on Jul 22 2009, 09:16 PM, said:

fishdoc, on Jul 23 2009, 09:52 AM, said:

Agreed. 2 ul for the sample and 2 ul for the water blank. Only use 1.5 ul to initialize the machine.

Hi fishdoc,

What I always do is to add 2ul and den close the cover and initialize the machine. After that is done, I just click on blank. This is alright, right? That was what my supervisor told me. Didnt know that I had to take a small volume to initialize first, den clean the machine, and add again to blank.

I'm sure that's fine as well. We have a central facility that has a bunch of equipment, including the nanodrop, and that's what their protocol said to do (how I do it). I don't see a reason that what you're doing would be a problem. However, like I said, I just use the machine per their protocol, I've never sat down and read the company's instructions for use.

[jiajia1987]

fishdoc, on Jul 23 2009, 10:29 AM, said:

jiajia1987, on Jul 22 2009, 09:16 PM, said:

fishdoc, on Jul 23 2009, 09:52 AM, said:

Agreed. 2 ul for the sample and 2 ul for the water blank. Only use 1.5 ul to initialize the machine.

Hi fishdoc,

What I always do is to add 2ul and den close the cover and initialize the machine. After that is done, I just click on blank. This is alright, right? That was what my supervisor told me. Didnt know that I had to take a small volume to initialize first, den clean the machine, and add again to blank.

I'm sure that's fine as well. We have a central facility that has a bunch of equipment, including the nanodrop, and that's what their protocol said to do (how I do it). I don't see a reason that what you're doing would be a problem. However, like I said, I just use the machine per their protocol, I've never sat down and read the company's instructions for use.

okay thanks~

[microgirl]

You should be blanking your machine with whatever your nucleic acid is in - ie initialize with the water then clean that off and blank it with EB buffer, TE buffer, or whatever your n.a. is in. Only blank on water if it's suspended in water.