no product on gel after restriction digestion - (Oct/03/2012 )
I have cloned a 500bp insert into a 7000bp vector, and I'm trying to confirm for correct insertion by use of a restriction digestion. Per sample I've done three reactions:
- uncut control
- digestion with AscI and NotI, which cut on both sides of the insert to check for insertion of the 500bp insert
- digestion with AscI, NotI and SmaI, which cuts twice in the insert to check whether it is the correct fragment that has been inserted.
The setup for the reactions was:
- 2.5ul 10x buffer
- 0.25ul BSA
- 0.5ul enzyme, 10U
- 500ng template DNA
- up to 20ul water.
Digestion at 37C for over an hour.
The problem is that when I run all three reactions on an agarose gel after digestion, I don't see any product at all. Not the expected digested bands, but also no undigested fragment. Also the uncut control is empty; ergo, all lanes are completely empty. Quantification by nanodrop reveals sufficient amounts of DNA in all samples, so I would expect to see at least some uncut DNA
Could anybody give me a suggestion on how to deal with this problem? If there is more information required, I will happily give it.
Thanks a lot in advance!
Maybe you are not adding enough Ethidium Bromide? Your samples could have run off the gel? Are your DNA fragments at a high enough concentration to be seen?
I agree with Smog187. Likely your gel running is the problem. Does your ladder show up on your gel? How is your gel stained? You should be adding water to 25 ul, if you are using 2.5 ul of a 10x buffer. Also, SmaI wants to be incubated at 25 rather than 37. I'd suggest that you skip the digestion steps until you can see a good band for your undigested DNA when you run a gel.
The other possibility is that you really don't have any DNA in the tube. How was the DNA prepared?
Smog187 on Wed Oct 3 19:12:06 2012 said:
Maybe you are not adding enough Ethidium Bromide? Your samples could have run off the gel? Are your DNA fragments at a high enough concentration to be seen?
My ladder does show up correctly on the gel, so this couldn't be the problem. The DNA should be of high enough concentration, but I'm now running a control gel with higher concentration of only undigested plasmid to check whether I can detect DNA in the samples.
phage434 on Wed Oct 3 21:57:10 2012 said:
I agree with Smog187. Likely your gel running is the problem. Does your ladder show up on your gel? How is your gel stained? You should be adding water to 25 ul, if you are using 2.5 ul of a 10x buffer. Also, SmaI wants to be incubated at 25 rather than 37. I'd suggest that you skip the digestion steps until you can see a good band for your undigested DNA when you run a gel.
The other possibility is that you really don't have any DNA in the tube. How was the DNA prepared?
The DNA was prepared by miniprep, in the same way that I normally get yields of a few micrograms/ul. Nanodrop confirms this high concentration, but nothing visible on the gel.
Thanks for your suggestion, like I said above I'm now checking higher concentrations of uncut DNA to see whether I can detect the DNA on my gel.