How to check digested products ? - (Apr/05/2008 )
I am trying to subclone a PCR amplified DNA fragement into vector PIVEX 2.4d. I have digested the PCR fragment and the vector with Nco1 and BamH1. Since I´m new to this,
can anyone tell me how can we check if the digestion is successful.
Also can anyone tell the right protocol for double digestion with NcoI and BamH1 (right amount of enzyme and DNA). I used the Qiaex II gel purification kit to elute PCR amlplified DNA.
Any suggestion would be highly appreciated!
To check your digest products, you would usually run them on a gel with ethidium to visualize to make sure you have the expected fragment sizes. (Run a ladder in one of the lanes so you can compare the band migration.)
Most digest reactions are 50µl total, with anywhere from 1-10µg DNA. (I've cut as much as 20µg in a 30µl reaction). But you can adjust as needed. You want to make sure that your buffer is added to the correct concentration (if you're using a 50µl total reaction with a typical 10x enzyme buffer, you would add 5 µl of buffer to the reaction.) Also, you want to make sure that your total enzyme amount in the reaction does not exceed 10% of the total reaction volume, since the enzymes are stored in glycerol, and too much glycerol will inhibit the reaction. Some enzymes require the addition of BSA (Usually supplied with the enzyme in 100x conc.), and if you should add it if either enzyme in your double digest needs it.
I don't know what brand of enzyme you are using. I use NEB, and their website is very helpful. You can find their double-digest recommendations at http://www.neb.com/nebecomm/DoubleDigestCalculator.asp
You should always add your reagents in this order:
buffer (and BSA, if required)
For NcoI and BamHI (using NEB reagents), NEB recommends that you use NEB Buffer 3 + BSA, at 37°C. For a 50µl reaction, I would use (added in the above order): 5µl of the 10x Buffer3, 0.5µl of BSA, 2.5µl of each enzyme, your DNA, and water to bring final vol. up to 50µl. I would incubate at 37°, ~2-4 hours to ensure completion. (This is most likely an excess of enzyme, but the more DNA in the reaction, the longer you should incubate).
Thank you very much for the info. I also use enzymes from NEB and as u said they recommend NEB buffer 3. But i guess i also have a question about about of DNA used. My PCR fragment after gel elution with 20 micro litre of buffer according to QIAEX II kit gives a total yield of about 0.8 microgram. Is that too low or enough for restriction digestion? Does anyone have experience of using QIAEX II kit? If so how good was your yield?
I use the Qiaquick gel extraction kit, which I think is essentially the same as what you're using. (I think the only difference is the filter in the columns). Did you let the buffer sit on the column for a minute before you spun it down? Sometimes that helps the yield. If you still have the column, you can try eluting again into a separate tube and see if you get more DNA. (I've done that too). Also, you don't have to elute with their elution buffer -- you can use water instead if you prefer. Did you use all of your PCR reaction?
0.8µg should be fine for a digest, but I would run it in a smaller reaction volume (30µl) if you will need to gel purify it again afterward. (The smaller volume may be easier to load into a well, so you won't have to split it up and your DNA will be easier to see.) If you do this, just make sure to adjust your reagent volumes... (3µl buffer, 0.3µl BSA, 1.5µl of each enzyme, etc.)