My single restriction enzyme digestion doesn't work - Digestion problem, no cutting. (Sep/18/2006 )
I'm doing a project which shows how RFLP method can be used to DNA fingerprint different goldfish varities. I can choose to do double digestion, but i want to start on single first and it seems it doesn't cut. All i see is smeared DNA. Each 2 lanes there is a different goldfish variety. Could it be that the sample size is too small to resolve the small fragments
1. I would improve gel quality, on the pic it does not look fine and to see differences in RFLP quality is not good enough (perhaps reduce voltage and gel percentage. For genomic DNA you get better results; or the buffer is too old)
2. If you just cut genomic DNA with 1-2 enzymes, normally you get only smear and no bands, as there are many cleavage sites, even with a 6 bp cutter.
3. I would use PCR-RFLP, then you have a known sequence where you can find suitable enzymes which produce bands of visible size (not to small). Problem is that you have to sequence at least one polymorphic part of the genome (e.g. ITS region), but it is easier and faster than the classic way with hybridisation etc. Other way: you combine it with RAPDs, i.e. random amplified DNA plus RFLP, there are approaches that worked.
4. Sometimes it does not work: In my RFLPs sometimes I had in my PCR amplified regions (with several retriction sites for the given enzyme) a sample where the enzyme was not working (which was not polymorphism as there where many sites). I never figured out why. So I started again with PCR or even DNA extraction, and then it worked.
Molecular biology is sometimes voodoo...
We'd need to see what an uncut sample of your genomic DNA looks like to see how much cutting is ocurring. It's certainly not none, but I would let the digestion go on for longer (4 - 6 hours perhaps).
Are you going to transfer the gel to a membrane and probe it? If you are, you need much less DNA. I've had Southern's work beautifully even with no apparent DNA on the EtBr stained gel.
If you're not going to blot it, I think you'll need an enzyme that cuts quite rarely to see disticnt bands. What is the GC content of goldfish DNA?
Ok, did it at 4 hrs and nothing even appear. I think I may know why there are no bands at all. My theory to why no digestion occur is because I overloaded the amount of restriction enzymes.
I have an average of 7.5ug of DNA in a 50ul volume of digestion solution. I am using the NEB enzymes, in stock, the concentration of units is 20,000U/ml, meaning that there are 20U/ul, by adding 4ul of enzyme as stated by "homebrew" I'm actually overloading the amount of enzyme required, and therefore the amount of glyercol in the solution is also more than required which could explain the smearing and enzyme being inactive.
By following "homebrew's" protocol, I'm loading 80U of enzymes to digest only 7.5ug of DNA in a 50ul volume.
Solution: Do a 2x dilution of the enzyme, making roughly around 5U/ul and add only 1ul of enzyme volume to the solution
So keeping the rest constant to homebrew's protocol, the components shld be as followed:
DNA sample - 10ul
Enzyme(with 2x dilution) - 1ul
10x Buffer - 5ul
10X BSA - 5ul
Sterile Water - 29ul
Total volume = 50ul
Incubation for 2hrs.
What do you guys think? Give me some feedback on my theory.
The Bam HI I routinely use is 10 units/µl, so the NEB's is twice that. With that in mind, I would do:
10 µl DNA
2 µl Bam HI @ 20 units/µl
5 µl 10X Buffer 2
5 µl 10X BSA (I assume you've diluted this from the 100X it ships as)
28 µl sterile distilled water
Your other point "...and therefore the amount of glyercol in the solution is also more than required which could explain the smearing and enzyme being inactive" is inaccurate. The enzyme is shipped in 50% glycerol regardless of the enzyme's concentration, so you're not adding more glycerol because your enzyme is more concentrated, you're just adding more enzyme.
You seem convinced the enzyme is "inactive". I am not so convinced. What does an uncut lane look like?
Also, as I asked before, are you planning on detecting polymorphisms by Southern blot, or are you just hoping to get a nice descrete banding pattern on an agarose gel? If it's the latter, I think you're going about it the wrong way...
Yea, its the latter, I'm trying to get banding patterns. How should I go about doing it. I'm using goldfish genomic DNA. Should I use polyacylamide gel for the detection ?
And before we continue on this topic, I would like to thank HomeBrew for your support in this. Thx, please keep feeding me with your advice.
Well maybe someone with more experience than I have with RFLP will have an opinion, but I think Bam HI will cut too frequently for your purposes. Double digests would surely exacerbate this problem.
If there are too many sites for the enzyme to cut, you'll get just about all possible fragment sizes, reulting in a smear of DNA on the gel. This is fine if you're intending to do a Southern, but is unlikely to produce a discreet banding pattern that you can compare across samples -- one smear looks pretty much like any other.
I think you'd need to use a rare cutting enzyme to get a discernable banding pattern...
i'v done RFLP before, but not with Bam H1 yet..are u sure u're using the specific restriction enzyme itself and its sure to give a cut..and if so,do u have any references of such a work done before or any such concentrations mentioned in any such works..pls go thru some references and u may find some help from dem..also,try reducing the quantity of ur DNA taken for digestion nd also try diluting ur restriction enzyme with the buffer provided and try preparing the mix avoiding sterile water..i had tried the same formulation and got gud cuts..
hope dis wil be of some help..
Thx for your reply, i tried using special cutters such as Sfi
5' GGCCNNNNNGGCC 3'
3' CCGGNNNNNCCGG 5'
I used this sequence to cut my genomic DNA, incubation requires the temperature to be at 50 degrees, I did so for 2 hours. I followed homebrew's recent volumes. And this is the result. Picture attached see what u can make out it. For the marker i used a 100bp ladder and it seems its too much, since i loaded 6ul into a 8 weller gel. The gel concentration is 1.5% and it ran at 120V for 45mins.
If I used too little of DNA, i fear the smaller fragments will not be clearly visible.