Thanks to Bitesize Bio reader, Muthu Arumugam for contacting us about some problems he has been having with restriction digestion and clean up of DNA.
I have boiled his query down to four main questions that are pertinent for most molecular biologists, so I hope that Muthu and everyone else can learn something from my answers.
If anyone else would like us to personally help with a problem or question they might have in their bio-life, be it technical, career-related or anything else, please be sure to drop us a line and the collective might of the Bitesize Bio will swoop in to help you in your hour of need (ok, we will try to help!).
So here are Muthu’s main questions and my answers:
Q: What is the shortest nucleotide length which can be precipitated by standard ethanol precipitation? I don’t want use the PCR product DNA purification columns. In such case after double digestion with XbaI and BamHI there is almost 12 bp of nucleotide released. If I precipitate this double digested vector (pBSKS) will it precipitate those small released 12 bp fragment also?
A: The answer to this question is: I don’t know exactly, but ammonium or sephadex might help you. The size of the DNA that can be removed by ethanol precipitation using various salts and ethanol vs. isopropanol has never been critically analyzed down to the detail you desire (as far as I could ascertain using my incredible google research skills). An overview of the different salts used for ethanol precipitation and how to choose has been described before by Nick in this article.
Ammonium acetate is used for precipitating DNA with removal of dNTPs, but a 12bp fragment is going to be significantly bigger. And determination of whether or not 12 bp is removed is difficult to assess with simple methods such as agarose gels. In this case there are two choices. One is to try it and let us know. It would be an interesting techniques paper or student project to perform an analysis of removal of various dsDNA sizes using the typical salts and alcohols over time and temperature of precipitation to see what the cut off for size removal is using this method.
Another option if you want to avoid commercial kits is the use of G-100 Sephadex. Sephadex separates DNA based on size exclusion. I would recommend the GE Illustra G-100 which excludes (so elutes) everything 25 bases and higher. This should work to trap a 12 bp dsDNA. However, a note of caution. Sephadex will not remove the enzymes or salts from your sample. The sample elutes in the same buffer you have it in, just without the smaller DNA.
Q: Will ammonium ions will inhibit the activity of XbaI and BamHI?
According to the book “Experimental Techniques in Bacterial Genetics“, ammonium acetate is actually the best choice for ethanol precipitation of DNA because ammonium is very soluble in ethanol and the salts are efficiently removed with a 70% ethanol wash. Ammonium acetate precipitated samples tend to work best in enzymatic reactions, the exception being for polynucleotide kinase reactions.
NaCl and NaAcetate are less soluble in ethanol so will tend to carry over more salt in the sample. However, if the sample contains SDS, NaCl is the precipitant of choice because it helps keep the SDS from co-precipitating with the sample.
In other words, if your restriction digest isn’t working, it is not because of the ammonium acetate.
Q: One more question is regarding the two enzymes BamH1 and XbaI . How many bases does it occupy in the DNA in search for the recognition sequence?I am trying to digest with BamHi and XbaI using a 73 bp fragment and am not getting the clones I need.
A: 1 base for BamHI, 2 bases for XbaI, and for other enzymes it can vary between 1-5 bases.
This answer took some searching but I finally found it in a great experiment performed by Fermentas scientists described here.
To summarize, PCR primers were designed with 1-5 bases added on next to enzymatic recognition sequences and were P32 labeled and used to amplify fragments using PCR. Restriction digests were performed, products separated by 10% PAGE and the % efficiency of cutting was determined.
You will be happy to know that BamH1 had 50-100% cutting efficiency with just 1 base next to the cleavage site and XbaI needed 2 bases flanking the cleavage site to achieve 50-100% cutting efficiency.
So if your fragment has >2 bp flanking the cut sites, the enzymes should be cutting correctly. To be safe, you can always extend the ends by 5-10 bp and you should be totally safe.
Q: Does phenol has any effect on DNA shearing? My DNA is getting sheared and I need longer fragments for my experiment. Can you help me out?
A: No, the phenol is not shearing your DNA.
The shearing occurs from vigorously shaking the sample during the extraction. For high molecular weight DNA, you want to be very gentle. Invert to mix with slow easy motions. Do not use a vortex or shake the sample. To isolate the DNA, after ethanol precipitation of the aqueous phase, I prefer to use the spooling method to retrieve the DNA.
Take a glass Pasteur pipette and use a flame to create a loop on the end. After the DNA is precipitated, if the extraction was large scale, the DNA will come out of solution all in one piece and appear white and filamentous.
Use the glass hook to reach in and grab your DNA.
Hold the DNA over the tube and wash it briefly with 70% ethanol by using either a 70% ethanol squirt bottle, or a pipette to gently rinse the DNA with 70% ethanol to remove any salt.
Let the excess ethanol drip off and then unhook the DNA into a vial containing buffer at neutral pH.
If the DNA is stuck to the hook (it is sticky), then go ahead and break off the end of the hook into the vial and leave it there. It won’t hurt the DNA.
Typically, large quantities of DNA prepared this way (>100 ug) will need to dissolve for several hours to overnight at room temperature preferably with rocking or rotating. This DNA will be very large in size and suitable for making large chromosomal libraries.
My other tip for keeping the DNA high molecular weight is to use wide bore pipette tips for aliquotting the sample. To prepare your own, use a clean razor blade and a sterile petri dish to snip off 1-2 mm off the end of the tip. This will make sure the DNA doesn’t break from mechanical shearing due to pipetting.
However, it is important to note that DNA prepared with silica columns is typically in the range of 20-50 kb and this is large enough for most molecular biology applications. DNA prepared using a salting out method does not require phenol and will be in the range of 100-300 kb as long as you handle the sample gently.
So don’t be concerned that your spin columns are destroying your DNA. That DNA is fine too. PCR and qPCR performs better when the DNA a bit more broken anyway.
Thanks again Muthu! I hope this helped, but if not please feel free to ask further questions in the comments. If anyone else has anything to add to what I have said, please feel free to get involved in the comments too.
And again, if you have a question or problem you’d like us to answer, please give us a shout.
PIPE PCR is a ligase-independent, restriction enzyme-free cloning strategy like SLIC (link to my SLIC article), SLiCE and CPEC. The PIPE method eliminates sequence constraints and reduces cloning and site mutagenesis to a single PCR step followed by product treatment. It is fast, cost-effective and highly efficient. The key step is designing the primers; one […]
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