I don't think that would be a practical approach. If you dephosphorilate only one of the digestions, in the other one you will still have a lot or re-ligation, because the fragments are both phosphorilated and will bind more efficiently.

Also, dephosphorilation is never 100%, so you will also have re-ligation of the other plasmid. Plus, vector-vector and fragment-fragment ligations. Even if you add extra restriction enzymes to further cut the undesired fragments, I fear you would still end up with a lot of crap in the ligation mix.

You would, of course, have some correct plasmids in the mix, but the question is if you have enough to find some in your colonies. I would definitely do gel purification in this case, sorry about that (I hate it, too).

Hi peter. The mentioned strategy is practical but you will getting into trouble when you want to select your desired ligated plasmid. dephosphorylation cant help you because finally you will have 4 products (1. intact expression vector 2. intact cloning vector 3. expression vector with desired fragment 4. cloning vector with undesired fragment). I prefer conventional method (extraction of desired fragment from gel then setting up ligation). 

I hope be helpful.

Hi Peter,

I agree with Mohsen, I would do the standard digestion, and then gel extraction of your desired insert and vector, treat your vector, set up proper ligations and proceed from there.  The minimal amount of time it takes to do a couple gel extractions is worth it to me to prevent downstream cloning headaches.  

As the others said, although tempting to save time (in theory - you'd use selection, and hopefully the CLO plasmid has different res cassette than the EXP one), the only sure way to REALLY save time is the long road.

I completely agree with the things said above.

Of course you can try and the desired event will take place, but you will get in trouble to identify it: you need a selection strategy, which only allows the correct clone to survive!

Thus, I also recommend to go the old “long” way.

Good luck, Marlen

If you need to get the clone ASAP, you should go the gel extraction route, and you don't even need to dephosphorylate your vector. You will not even loose time, as you need to run a gel to verify completeness of digestion anyway. So it comes down to gel extraction (15 mins with a kit) vs dephosphorylation (15 minutes, but then you must inactivate the phosphatase for another 15). On the other hand, I kind of like your idea of just mixing the two reactions, so I would probably give it a try. Especially if you will be repeating similar subcloning over and over.

Good luck and let us know if it worked.

Darius

Hi Peter

I am agree with Darius views. There is nothing wrong in purification or gel extraction. There will be hardly 20% loss of your DNA but you will be assured with good quality without much impurities. We usually skip dephosphorylation step and we get good cloning results under suitable selection pressure.

Good Luck 

I've done this a lot and never bother gel purifying or phosphatase treatment. You must be sure that you get good digestion, any residual uncut plasmid will skew the results. I usually just mix the DNA's and then transform a bit of the DNA after digestion but without ligation to see what the background is alongside of the transformation after ligation. If the number of colonies after ligation is higher then all worked well. Just be sure to screen a number of transformants and not just 2 or 3. I usually do 12 or 24. 

Hi Peter,

I agree with the above mentioned strategies. But in case if the gel extraction method is not feasible for your cloning, I would suggest the following approach. If you know the sequence of the insert and the sequence of expression vector, you can probably go for a PCR approach to identify the clones with your desired insert in the expression vector.

All you have to do is to design your forward primer from the expression vector and design a reverse primer from the insert. After you perform the cloning with dephospho rylation approach, you can pick single random colonies and suspend them in 10ul of water. Take 1-2ul of this sample for the PCR reaction. 

If you have positive bands on the gel with expected product size you can be sure that your cloning worked. You can even take leftover water samples with bacterial clones and go for maxi-preps. I have been doing this kind of cloning when my insert size is less than 100bp which makes it difficult for performing gel extraction. Hope this might help!!!