Hi Fan,

The restriction itself looks good - however if you put 20 µl of the linearized vector on the gel your vector concentration is really low.

However, I think to answer your question you have to give some more information.

How much DNA have you used for the digestion?

What do you mean with recovery? Did you purify or precipitate your DNA after digestion. And if yes which method have you used?

I think with some more information I or another research might be able to help you.

KR Dörthe

Regarding the low recovery rate of linearized vectors after enzymatic digestion, this depends on the recovery kit used. You can also set up a control group without enzymatic digestion for comparison. Both digested and undigested plasmids will appear in the same band, so the band thickness and brightness are not related to the enzymatic effect. Finally, for experimental condition optimization, ensure that ddH₂O is not contaminated, as contamination of components can also affect enzymatic activity.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

[email protected]

You haven't provided enough useful information, how much DNA did you start with and how much did you end up with? To say you loaded 20ul is meaningless, is it 20ul from a total of 100, or a total of 1ml or a total of 1L?

Having said that, each lane looks like it has around 20-50 ng loaded (very rough guess) but if you know how much ladder you loaded you can calculated a reasonable estimate based on the mass of the band at the similar size.

Dörthe Andrea Kesper

Thank you very much for your reply!

Here are more details about my protocol:

I used gel recovery to purify the linear vector. The amount of plasmid digested was 1 μg, and the concentration of the linear vector after digestion was 30 ng/mL. The recovery rate refers to the percentage of the linear vector obtained and the plasmid before digestion.

• Amount of DNA used for digestion: I used 1 µg of plasmid DNA for the restriction digestion.
• Reaction conditions: I used EcoRI from Medchemexpress (HY-KE7014), with the recommended buffer, and incubated at 37°C for 1 hour.
• Volume: The total digestion reaction volume was 50 µl.
• Post-digestion recovery: I ran 20 µl of the digestion mix on a 1% agarose gel (as seen in the image). I excised the linearized band under UV and purified it using a gel extraction kit (QIAquick Gel Extraction Kit). The final elution volume was 30 µl. After measuring the concentration with a NanoDrop, I noticed the yield was very low, which led me to ask this question.

Thanks again for your time and help!

Michael J. Benedik Thank you very much for your reply!

I have added some details of my protocol in the comment section.

By “recovery rate,” I meant the percentage of linearized vector obtained relative to the initial 1 µg of plasmid input. Given the concentration and elution volume, the total recovered DNA was around 900 ng, so the recovery rate was roughly 90% — but the concentration feels low for downstream applications like ligation.

Thanks again for your help!

Hi Fan,

a recovery rate of 90% is pretty good - I don't think you will get a better recovery rate!

To get more vector - you can digest more of the vector - digest 2-3 µg of vector (usually this should be no problem even with 1 µl of the EcoRI restriction enzyme, because it cuts really well and the restriction enzymes are normally highly concentrated, however to be on the save site you can digest for 2 h - no problem as EcoRI has no star activity).

When I see it correctly you can even use concentrations up to 10 µg with QIAquick Gel Extraction Kit (however I think you don't need to do this).

I would also recommend a digestion volume of 20 µl - this is no problem to do and you can put the whole ligation mix in one well of your gel (even if the wells are not very big) - this will reduce the amount of gel you are working with subsequently.

When you use larger amounts of your plasmid be careful with the size of the gel slice (do not overload the column with gel as this can greatly decrease your recovery rate). If you gel slice is big use two columns.

And be aware if you cut out your DNA under UV light - be fast cutting it out, because UV light is harmful do your DNA and decrease transformation efficiency fast and might introduce mutations (check here: https://bitesizebio.com/10264/turn-away-from-the-uv-light/).

Another remark: Depending on the size of a vector ( I am not sure how big your vector is) 25 ng-50 ng should be enough for a ligation and cloning.

I usually used 25 ng vor vectors 3-5 kb and 50 ng for vector up to 10 kb. So with 900 ng of vector you can perform several ligations.

Regarding using the Nanodrop - after purification with columns the results might not look optimal, because you might have some chaotrophic salt in the purified product, but this should not interfere with subsequent applications.

Beside using the photometer I usually also estimated the amount of DNA simply by comparing my DNA band with the ladder (were you know the amount of DNA for a certain band) - to see if this is in the same range.

And just for the record - I am not sure if I understood this right - however the remark from Qui "Both digested and undigested plasmids will appear in the same band" sounds like you get the same band for digested and undigested plasmid (maybe I got something wrong here) - however this is not the case.

If you have undigested plasmid you will see 2-3 bands.

1. supercoiled vector - supercoiled vector runs fast in a gel and will run below your digested/linearized vector.

2. nicked/relaxed vector - this runs slow and will run above your digested/linearized vector

3. linearized vector - will run obviously at the same hight as your digested/linearized vector - however depending on the quality of your plasmid prep this band might be faint.

I also do not see any sense in it using this as control - at least not in your situation as your vector is clearly cut.

When you see more than one band in a digestion your digestion is incomplete (what I would not expect with EcoRI - at least in my hands this was one of the most reliable retriction enzymes I worked with).

You might do this control if you suspect that your digestion did not work at all and you are not sure about this - but again this is not the case with your digestion.

And finally - as you cut out your vector band even incomplet digestion should be no problem.

So this was a lot but I hope this helps

Dörthe Andrea Kesper Thank you again for the detailed explanation provided, this is very helpful!

Regarding on the bands for plasmid, I confirmed with Qiu via email, and he/she explained that the digested plasmid and the undigested plasmid would appear in different positions in the same lane, and the thickness and brightness of the recovered bands could indicate the DNA content. If the corresponding band of my linear vector is bright enough, it means that the digestion reaction is effective and successful. As for the final recovery efficiency, it is related to my recovery kit.

Hi Fan,

for me this is a strange explanation - as I told you it might be that with the undigested vector you will have a band at the exact same position as your linearized vector (if there are double strand breaks in the vector due to damage) - so this explanation is also not correct .

Here you can see how this would look like :(https://www.coursehero.com/tutors-problems/Biology/26640961-Following-electrophoresis-of-the-two-pGlo-mini-preparation-samples-bo/) - please check this out. So not all of the undigested vector willl appear at different position (lane 4 you have clearly a band at the same hight as in lane 3 and 5 - even if the plasmid is not digested, lane 1 is marker).

And I don't understand why they should appear in different position in the same lane? Because you sure should use two lane for two different DNA samples (however maybe that just the wording)?

The brightness has nothing to do with your restriction efficency itself - when you have only one band which is not so bright at the expected hight of your linear vector, it would be still complete digestion. It would just mean that you did not have a lot of DNA in the reaction in the first place.

And if you see more than one band this would indicate incomplete digestion (when the band of at the expected hight is brighter than the other bands you can conclude than more vector is digested than not - however it would still be incomplete digestion what is bad (and I would not consider this as sucessful)) and should not occur with an enzyme like EcoRI.

Please have a look at the picture following the provided link - for a succesfull digestion it shoul look like in lane 3 and 5 (lane 1 marker) and nothing else (if you have only one EcoRi restriction site in your plasmid)

However, your vector very clearly is completly digested - and you should not worry about incomplete digestion as long as you see this picture with your digestions.

What is correct is that the thickness and the brighness of recovered band will indicated the amount of recovered DNA.

Regarding your final recovery rate this is surely related to your kit - however there are also other factors at play like the amount of DNA, the amount of gel, the amount of elution buffer which can strongly influence your recovery rate (however what you showed seemed pretty good)

And be always aware that your final success in ligation is strongly influenced by the time you exposed your DNA to UV-light (indepenent of the recovery rate.

I am sorry for this lenghty explanation - maybe it was just because of the wording that some things sounded weird, however I think it is really important to understand these things and the different conformations a plasmid can have and how an effective digestion looks like if you are working with plasmids.

KR Dörthe