Hi Thomas,

We are used to linearize large amounts of plasmid (> 1 mg) using restriction enzyme digestion since we prepare run-off RNA transcripts for NMR purpose.

The low yield problem that you describe (i.e. max 20 ug out of 50 ug) might be due to overloading of the (small?) spincolumn (max binding capacity is usually 20 ug). Or do you use several small columns?

To save on restriction enzyme we always make a small enzyme dilution series for a given DNA concentration prior to large scale digestion and in some cases we can go down to digest 1 ug of plasmid in 40 ul using 2 units of enzyme in an overnight incubation. 

To obtain the maximum yield, we load the digestion reaction onto an HR anion exchange column (1 ml) connected to an FPLC and elute the DNA in a small volume which we than concentrate further by ethanol precipitation. But we have digestion reactions of 50 - 200 ml easily but likely this can be scaled down.

I hope this helps.

Best,

Frank

RE digestion can be done to linearize large amounts of plasmid.

You can please refer to the following links to get certain valuable details:-

1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC213547/

2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816399/

For NEB  Restriction enzymes, I unit is the amount of enzyme  required to digest 1 ug of DNA at 37 degrees for 1 hour. either you want to use 50 ug of your linearizing enzyme or you can try optimizing RE digestion by adding say 2.5 ug DNA and let it digeste for 2 hours and then if it works you only need to use 20 units of  enzyme to linearize 50 ug of your plasmid. By the way, what organism are you using? Do you really need that much DNA to transform your cells? Seems like a lot. Maybe you can look at your electroporation conditions and see if you can improve the efficiency of your transformation.

Dear Thomas,

did you already consider a potential substrate inhibition? How much is the concentration of plasmid in you assay (µg/µl)? For the assays for which you state only a 40 % digestion success, were these test-assays in the recommended substrate concentration range?

You might like to increase the volume, but instead of using more restriction enzyme, it might be possible to extend the reaction time to e.g. overnight. For NEB-enzymes, you will find suitable enzymes for extended digestion by the following link:

https://www.neb.com/tools-and-resources/usage-guidelines/restriction-endonucleases-survival-in-a-reaction

Good Luck! and best regards,

Christian

Thanks for your suggestions everyone.

I don't have problems with inefficient digestion, but low (DNA) yield after column clean up. 2 different kits. I usually use them for stbl3 mini clean up and they work just fine. Using my 50µg (no matter how well digested) my yield is like 20µg, and that only after several elution steps and 2nd round clean up of the flow through.

I need large amounts to EP different human cell lines for stable expression.

So my main problem is the low yield after clean up. Secondly I want to save on RE, and yes increasing time was definitely my way to go.However, I read in a guide (and it is stated on manufacturers protocols (incl. NEB), you shouldn't use more than 1µg per 20/50µl. The guide says increasing volume  (say 20µg/400µL) leads to low efficiency.

It would be great if someone had experience with what I am trying to do, and has a working protocol for me.

Cheers,

Thomas

In my experience cutting DNA with NEB, fermentas , or other major brand enzymes, It is not an issue to add more enzymes than the protocol recommend (remember that is based on the molar concentration of lambda phage dna). Typically I have digested 5-7 kb vector with two different restriction enzymes and 40 Units in total in a 50 ul final volumen (aprox 3 times molar concentration than your vector) for 2 hours, all clones correctly ligated. So yes I think you can cut more than 1 ug of dna  in low volumenes increasing time of incubation.

On the other hand for your purification if :

1. you elute in H2O for electroporation you have to have adjusted the pH (7-8) of this solution for efficient elution. 

2. Do not overload the volumen more than recommended for your spin columns clean up.

RE digestion is the best way to linearize the large plasmid DNA

That much amount of DNA for transformation, seems unusual! Anyway, you can go for phenol chloroform extraction instead of column clean up , so that there would be less DNA loss.

I also suggest that you go for phenol chloroform extraction to avoid column clean up to maximize your DNA concentration.

Hi Tomas,

you digest the plasmid with any one of the restriction enzyme to make it linealized.

all the best

You can Phenol-chloroform extraction 

Doing a column clean up after digestion should not be a problem; however, most standard columns have a binding capacity of only 25ug (miniprep columns and most clean up columns are only 5ug).  If you're loading 50ug, then your yields will at best be 50% or worse, which is what Jose was inferring, so you will have to spread them out over multiple columns and concentrate if needed, or find binding columns with higher binding capacities or what others have suggested above (phenol/chlorofom,, etc)

Another option is to incorporate an easy enzyme site in your plasmid so that you can cut more easily (and cheaper) your bulk preps but the above conditions still apply.

Hi Thomas,

We are used to linearize large amounts of plasmid (> 1 mg) using restriction enzyme digestion since we prepare run-off RNA transcripts for NMR purpose.

The low yield problem that you describe (i.e. max 20 ug out of 50 ug) might be due to overloading of the (small?) spincolumn (max binding capacity is usually 20 ug). Or do you use several small columns?

To save on restriction enzyme we always make a small enzyme dilution series for a given DNA concentration prior to large scale digestion and in some cases we can go down to digest 1 ug of plasmid in 40 ul using 2 units of enzyme in an overnight incubation. 

To obtain the maximum yield, we load the digestion reaction onto an HR anion exchange column (1 ml) connected to an FPLC and elute the DNA in a small volume which we than concentrate further by ethanol precipitation. But we have digestion reactions of 50 - 200 ml easily but likely this can be scaled down.

I hope this helps.

Best,

Frank

Thanks everyone for further input!

My clean up columns have a capacity for 100µg, so that shouldn't be a problem. Also, I re-loaded the flow through.. and so on.

@Frank: Thanks for your answer, finally someone who actually does this kind of thing. Do I get you right that you are digesting up to 1µg in up to 200µl? So do you think digesting 50µg in in 200µl shouldn't be a problem then? Just in terms of volume? I will  determine RE concentration for several hrs/overnight then.

Hi Thomas,

Indeed, the binding capacity of your columns should be sufficient to recover the majority of the material....if the problem persists, you might try to let the DNA bind to an anion exchange matrix, which is more robust than binding to a silica matrix.

Concerning the DNA concentration, I usually end up taking 1 ug of DNA per 40 ul or 50 ul. In my experience the concentration of the DNA is one of the critical factors to obtain complete digestion in combination with the reaction time. You wrote that you use a FastDigest enzyme? I don't know if there is also a conventional variant of the enzyme that you use? Maybe from another supplier? I'm not used to use FastDigest enzymes because the company doesn't give any unit information of the enzyme.

I can't guarantee that it works, but I think that if you indeed scale the volume to 2000 ul (or 2500 ul) for 50 ug of plasmid (and use the enzyme volume for 20 ul reaction) and elongate the reaction time you'll obtain full digestion. 

Please let me know if it worked, I'm quite curious.

Best,

Frank

Hi Frank,

I just saw I didn't read that right: you are using 50 - 200mL.

So just a final question the 2 - 2.5mL just in 2 Eppendorfs Cups or several small smaller volumes? I.e. does reaction volume matter at all in your experience?

Cheers,

Thomas

Hi Thomas,

Indeed...that's how large our digestion volumes can be :-)

The volume doesn't matter as long as all the buffer components are 1 X concentrate. Two eppendorfs of 1.25 ml should be fine!

Best,

Frank

So I set up a little test digesting 20µg in 1mL with the Fast Digest Enzyme and it worked just fine: 1µl RE cuts 1µg/5minutes, so I used 2µl RE for 20µg/1h and got complete digestion.

Measuring my DNA right after the digestion (Qbit) gave me ~15µg, so some 25% loss, somewhere somehow. I could live with that. But my real problem is the clean up. Using the silica columns, with 2 rounds of elution yielded me a total of 3.3µg DNA, i.e a yield ~22%. It's really too strange. I will try my luck with anion exchanger columns now, if people are willing to send me samples...

For the record: I tried several things toincrease yield, including long elution time on the column, Elution at 37°C and 50°C, several rounds of elution. Admittedly all H20, but still.

I guess I'll do some more troubleshooting today. Waste some plasmid. Some time, too..