I usually clean between sequential digests. If you go to the double digest NEB and you check the detailed protocol, it gives you a great suggestion.

Try to follow this one, what they say. And I think that your incubation at 37degC instead of 25degC can cause the trouble. And I would go 1h digest each.

Hey,

i don´t know how you purify your DNA between the two restrictions. I had not compatible buffers in a few experiments, too. For me it worked well to precipitate the first restriction with Ethanol at -80°C for an hour, centrifuge, dry the DNA, resolve in a appropriate volume water, add your buffer and second enzyme.

Maybe thats an option for you.

if you are using small amounts of dna it increases yield if you add glycogen ot tRNA as a co precipitant which brings down more dna and also make the small dna pellet stick better to the bottom of the tube . Since both enzymes are a bit fussy about what buffer they cut in make sure you wash away all of the salt after ethanol purification.

When you are using two different buffer you have to purify between each restriction. Whether you purify by phenol-chloroform and precipitation or on column like PCR clean up is up to you.

1. This also depends on the amount of starting material (DNA) you used. Don't forget, each purification (ex. gel purification) you will lose certain amount of DNA. If you use tiny amount of DNA from the biginning for digestions and purification, you might not see the final product when you run them on the gel. Check how much DNA you used.

2. I double check 'double-digestion' for these 2 restriction enzymes. And yes, 'sequential digestion' is recommended (see attachment). But, one thing interests me is that 25oC is suggested for SmaI digestion reaction (see attachment).

Hi Paul,

Do you know the mechnism that tRNA can bring down more DNA?

Not really Yau. It is a nucleic acid so becomes less soluble in alcohol/salt and I always thought that it takes a certain minimum amount of insoluble nucleic acid to aggregate and form a particle large enough to spin down rather than just remaining in suspension but I never saw a reference for this other than Maniatis 1982 manual which does not give a mechanism and often linear acrylamide is used these days as an alternative

bw

Paul

Thanks for the explaination, Paul. It makes some sense. However, in the case, wether we can add some short DNAs (ex. sonicated salmon sperm DNA fragments), instead of tRNA.... RNA materials can be easily degraded.

yes I and think glycogen or linear acrylamide have the advantage that if the dna is used for pcr then carrier dna might amplify and that might be a worry. Perhaps long polybase oligos like A25 or c25 would be safe and will precipitate ok but yeast tRNA (crude) was always a cheap option from Sigma and possibly even in degraded for may be long enough to precipitate

Hi Paul,

Ok, I just found this info online (see attachment). People do use 'yeast tRNAs' for Carrier or co-preciptant. I was wondering where we can purchase tRNAs from. In the earlier email, the mentioned Salmon Sperm DNA fragments (sonicated) is also used for Carrier, but I don't think that it has been used as co-preciptant.

https://www.gbiosciences.com/Buffers-Reagents-Chemicals/Yeast-tRNA

Hi Paul, interesting!! I learned something new today. Thanks for your answers.

yes I only came across salmon sperm dna as a carrier in southern blots to minimise non specific binding of the probe, Always a pleasure to chat Yau

bw

paul

I usually clean between sequential digests. If you go to the double digest NEB and you check the detailed protocol, it gives you a great suggestion.

Try to follow this one, what they say. And I think that your incubation at 37degC instead of 25degC can cause the trouble. And I would go 1h digest each.

Hi

You can do digestion sequentially .

Good Luck !!