How to check double digest efficiency of a linear PCR product? - Molecular Biology (Dec/01/2006 )

Hello experts,

I have a linear PCR fragment (1.4kb long).
It has a BamHI and EcoRI sites at its two ends (with 4 bases flanking their ends)
How do I confirm if my double digestion (in EcoRI buffer) has worked? without actually going through the ligation-transformation-miniprep-digestion protocol.
Any suggestions are very welcome.
thanks

i would like to check the linear PCR fragment by another enzyme digestion which will cut the fragment for example as 500 bp and 900 bp or something like that.

or if u just worried about ur double digestion then if u use the standard procedure ..........then no need to be worried.
http://www.neb.com/nebecomm/DoubleDigestCalculator.asp? u can try this
but BamHI may have star activity in EcoRI buffer

Thank you.
But how will digesting with another enzyme help me detect this, because both my sites are at the very ends of the linear PCR fragment. [4bp-RE1 site - 1.4KB PCR product - RE2 site-4bp].
Digesting with a third enzyme doesnot seem to resolve whether this fragment is cut at both ends or not.

I would like to know if there is something I am missing.

I did my double digest using the NEB double digest calculator.
Thank you.
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Pray to the Gods of Molecular Biology, specifically the Goddess of Ligation. Make a sacrifice of 3 days of your life. Sacrificing human lifespan is usually the ticket to situations like this.

Ie. I know of known way to test for such a digestion. And in all probability neither does anybody. Hence, the lively forum of people coming here to seek advice for failed ligations. Plasmid contruction is tricky precisely because it is a long series of step with few points at which you can test your DNA to determine if all is going well.

Pray to the Gods of Molecular Biology! Pray!

Here is some information from this Promega website..

http://www.promega.com/guides/re_guide/chaptwo/2_6.htm

The addition of upstream bases to PCR primers is not the only method used to improve digestion efficiency. A number of protocols have been proposed to improve digestion including proteinase K treatment to remove any thermostable polymerase that may be blocking the DNA, end-polishing with Klenow or T4 DNA Polymerase and the addition of spermidine. However, none of these methods have been shown to improve cloning efficiency significantly (4,5).

An additional drawback to the incorporation of restriction enzyme sites in PCR primers is that it can be quite difficult to resolve digested PCR products from those that remain uncut. This can be overcome by the addition of fluorescent tags at the 5´ ends of the primers prior to PCR. This allows identification of products that have been cut successfully because the label is lost upon digestion (6).

An alternative method that has been used successfully to improve digestion of PCR products is to concatamerize the fragments after amplification (1,5). This is achieved by first treating the cleaned up PCR products with T4 Polynucleotide Kinase (if the primers have not already been phosphorylated). The ends will already be blunt if a proofreading thermostable polymerase such as Pfu( was used or may be treated with T4 DNA Polymerase to polish the ends if a non-proofreading polymerase such as Taq( was used (5). PCR products are then ligated with T4 DNA ligase. This effectively moves the restriction enzyme sites away from the ends of the fragments and allows efficient digestion.

You can easily test ligation efficiency of cut PCR products by ligation and running a gel. I would recommend cutting the PCR product with each enzyme individually. Then, ligate the results with normal (not quick) ligase and heat kill the results. Run the product on a gel and look for the double length product. The amount of double vs. single length product gives a direct measure of the efficiency of cutting and ligation. If you do this with both enzymes and achieve decent results, then you can be reasonably confident that the PCR product will be cut efficiently when cut with both enzymes.

Cleanup of PCR reactions prior to cutting is essential -- proteinase K works well to eliminate the enzymes, and column cleanup following this works well. There is wide disagreement on the source of the difficulties in cloning digested PCR products.

hi brami,

i think after double digest run ur digest on a 5-6% PAGE. Load both ur digested and un-digested products, u will see the undigested product runing slower than ur digested one if not then its not digested. if u want u can also include the single digests from each enzyme which will lie in btween ur undigested an digested, and also will tell which of the two enzymes has worked. 5-6% PAGE shud suffice as it resolves as close as 1-2 base pair. u cud either stain by Et-Br or silver stain. hope it shud do the trick (save time and effort by avoiding ligation, re-digestion etc)......!
gud luk........!

why you want check digestion efficiency
generally, 2 bp flanking is enough for efficient digestion using BamHI and EcoRI.
while I have suffered a problem and later found something wrong with ordered primer (not the same sequence as I ordered)