Dilutions confused me here. . .
For this typical procedure, I make 5ng/uL (nanograms per microliter) of DNA from a stock solution.
From the stock I have to dilute it 1:10 (10uL DNA and 90 buffer) to make it 50ng/uL
Then dilute that again 1:10 (this time 10uL DNA, 70 buffer and 20 tracking dye) to make it 5 ng/uL.
I then use this solution to put as samples on a gel for electrophoresis, and each sample has to be 50ng/uL (so I pipette 10uL from the 5ng/uL dilution.
The problem that confuses me is that now I need to do 12 samples. The 5ng/uL solution is only good for 10 (Because I use up 10uL for each sample and the dilution is 100uL) I am confused as to how to accommodate a slightly larger dilution without messing up the concentration. The more I thought about it, the more confused I got. I was thinking of simply adding 20 more uL of buffer into the 5ng/uL dilution, but wouldn’t that dilute the DNA more? Yet I’ve done things like that before when I looked back at older experiments and now I’m confused as to why the concentration of DNA doesn’t get more dilute even though I add more dye here and there or more buffer here and there.
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@gailcalled Well, two questions: First, given the 5ng/uL DNA dilution I described I’m used to making, how do I make it slightly larger for 12 samples this time (20uL of something needs to be added to the entire dilution)
And second, doesn’t the concentration of DNA, 5ng/uL, become more dilute if I make the solution bigger?
Seems to me you simply need 20% more of the sample DNA and diluent and you are good to go. So if you need to work with 10 µL of sample, you’re going to need 120 µL of diluent instead of 100, and 600 ng of DNA instead of 500 ng.
But my stock solution comes at 500ng. I can’t change that. It’s what we got. I know I need 120uL instead. But is it okay if I just add 20uL of whatever to the dilution? As long as I don’t touch the amount of DNA I transferred from the 50ng/uL dilution, there shouldn’t be a problem, right?
I do not know how sensitive the tests you are going to run are to concentration levels, but if you keep the sample volume the same but boost the diluent by 20% you’re going to have a concentration level 20% lower than it should be. Unless you know for certain that a sample 20% less concentrated will not harm the test results, a safer method would be to prepare 2 batches, make the required 12 tests, and toss out the unused portion of the second batch.
I was thinking of using 2 batches. But I did it anyway with the extra 20% diluent. At least I’ll see what happens. See, I got confused because in previous experiments I have added 10uL of extra diluent for electrophoresis to 10uL of sample, and it still came out at 50ng. So maybe an extra 10 will be negligible still.
Hmm, so you have a few errors in the question that were throwing me off, but I think I get it. So, what experiment are you doing? Electrophoresis of DNA is usually not highly sensitive, a difference of 20% will be noticeable but won’t result in the results being unusable, probably. Now, the gel is sensitive to the amount of DNA, not the dilution, so diluting it with the running dye doesn’t affect it if the original dilutions are done correctly, though if you run one well with 50 ng of DNA and one with 40 you will see a difference (so, if you do a second gel now with the full amount from two batches you’ll see a difference).
Now, from your last line, “I have added 10uL of extra diluent for electrophoresis to 10uL of sample, and it still came out at 50ng”, again that’s because you are loading a consistent mass of DNA, even if you dilute it after taking the proper amount. But in this case you don’t have enough to do it, since you’ll need a total of 600 ng (50×12) and you only have 500 in your dilution. So, if you want to do 12 wells with 50 ng each, there’s no getting around making more. However, I would suggest checking carefully how much mass you loaded in previous experiments, not concentration, as that may reveal something that you did differently, or that you just loaded 41.67 ng before (500\12).
@BhacSsylan Someone told me I just need to add more than 10uL DNA from the 50ng/uL dilution, which made me hit my forehead because now I get it. So if I wanted to make 120uL of 5ng/uL as a 1:10 dilution, I would have to mix 12uL of 50ng/uL DNA with 74 and 34 buffer and dye respectively, right? I’ve been told to do 140uL now though, because you should always have some extra for pipetting error. So then. . 14uL of 50ng/uL DNA with 88 and 38 buffer and dye respectively.
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