DNA concentration no spectrophotometer - (May/24/2015 )
Not sure what to do. I had 150ul of PCR reaction which I used QIAquick-pcr-purification kit on. Instead of 50ul of EB, I used 50ul of nuclease free water. My next step is to do restriction digest however, I have discover that the spectrophotometer doesn't work. My question is how do I figure out the concentration of the DNA so I know how much enzyme to use?
In order from more precise to less precise:
1) Is checking them on a fluorimeter an option?
2) Run an aliquot of your purified PCR product on agarose gel, alongside a lane of a quantitative ladder (i.e. Lambda DNA digested with HindIII, or some other proprietary quantitative ladder). Then capture an image where the bands aren't saturated and do a densitometric analysis of the bands using the ladder as standard.
3) As option 2, but view it under a transilluminator and eyeball the quantity of DNA comparing it to the ladder bands
4) Most restriction enzymes do not require an exact amount of units to be added. You could check that and, if overshooting is possible, then just use a rule-of-thumb amount of enzyme (for an eyeball estimation, I'd be surprised if you had more than 2ug of PCR product in the initial 150 ul).
Back when I was a PhD student we didn't have a nanodrop, and I used to check purified PCR products on a gel prior to ligating them. Nowadays I actually use a fluorimeter, it's more accurate than a nanodrop at the low DNA concentrations that come out of PCR purifications.
I never check the amount of DNA in a restriction digest. Almost always 1 ul of the enzyme (or even 0.5 ul) will over digest any reasonable amount of DNA in an hour. You are over thinking things if you are calculating it. A gel would be a good idea, but mostly to verify that you really have the DNA you think you do.
This is a small lab outshoot, we're lucky to even have the spectrophotometer.
After the PCR reaction, I ran an 1% agarose gel using 1kb plus ladder to verify that I had the DNA band I wanted and I did. I plan to do .8% gel once I've done the enzyme digest. Another question, since I have 50ul, would it be better for me to just use 25ul since the total mix (DNA, enzyme, buffer & water) is suppose to be 50ul?
In general only a small fraction of your restriction digest reaction should be your DNA. Enzyme inhibitors such as Gu-HCl and ethanol are common DNA contaminants, and will prevent digestion. When most of your digestion reaction is water, you dilute these contaminants.