Hi,
I am trying to digest my insert of 8.5 kb long with NdeI and XhoI (both obtained from NEB) but the insert gets destroyed after the digestion. I do not see anything in agarose gel. Kindly suggest some remedy for this.
I use following recipe for the digestion:
Ingredients Final concentration
1. DNA (Insert): 1 ng/micL
2. Cut Smart Buffer: 1 X
3. XhoI: 0.16 Unit/micL
4. NdeI: 0.16 Unit/micL
the final volume is made upto 120 micL with nuclease free water.
The mixture is incubated in 37 degrees Celsius water bath for the period of 3 hours.
I'm not sure how much of your DNA you are adding to the digestion, but based on your concentration of insert I doubt it would be visible on a gel. I think 10 ng of DNA is barely visible as a faint band and a good band requires at least 50 ng or more.
Are you digesting a PCR product or a plasmid or a purified insert?
I think that Michael J. Benedik is correct but additionally are we sure that there is an insert present and that the enzymes are not just opening empty vector ?
Hi Michael J. Benedik and Paul Rutland
Thanks for your prompt response. In my case, I am trying to clone 8.5 kb DNA insert to pFLAG-CTC vector (directional cloning). I digested the vector with these two enzymes but I am facing problem only in digesting my 8.5 kb DNA insert. The initial concentration of DNA (gel purified PCR productI am using is 10 ng/ul. In the digestion mix, the final concentration of DNA is 1 ng/micL.
Hi Shantanu Tamuly
You didn't answer the important question, what is the source of your insert? Are you digesting it from a plasmid, or is it a PCR product, or something else?
I think the main problem is not enough DNA, so the first suggestion would be to significantly increase the amount of DNA you are using.
Hi Michael J. Benedik
I am using the agarose gel purified PCR amplicon obtained from the genomic DNA of Salmonella.
In restriction digestion reactions, it is advisable to use high concentration of template in the restriction reaction. You can then run the restriction reaction on the gel. If you can see the expected size of the insert on the gel, you can gel purify it and clone the purified product in a vector e.g pJET. Once the insert is cloned in a plasmid vector, you can re-amplify it from the plasmid using primers specific for your insert and in that way, you can high concentration of the insert for any upstream work e.g sequencing etc
Shantanu Tamuly Is it really necessary that you see it on the gel?
I have 2 recommendations:
1) You have already gel-purified it, yes, the concentration is really really low. But you can just digest it and directly use it in your ligation / downstream applications.
2) you told us that it is a PCR product; so repeat your PCR once again using the gel-purified 8,5 kb insert with more than one reaction (use 8 samples e.g.) and check 5 µL of it on the gel. Then, the amount should be enough to see it on the gel.
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