Sal I cloning problems - (Jan/24/2008 )

Hey there,

I feel like I've got a pretty good grasp on cloning these days but im not sure where to go next on this one. I'm cloning 3 separate genes (1.5 kb each) into the Xba I/Sal I site of my vector (6 kb) and only got the same number of colonies on my test plates as i did on my vector only plate (indicating no ligation of my inserts). I think the vector preparation is ok because i got a low number of background colonies (3). It's not the proximity of the cloning sites because they are about 80 bp away from each other. I've also tested the efficiency of each enzyme on digesting the vector. I performed separate Not I/Xba I and Not I/Sal I digests, which showed that Xba I and Sal I are working perfectly fine (on the vector at least). All other ligations and transformations i performed at the same time (some in the same vector but using the EcoRI site instead) worked perfectly well so the problem is specific to these restriction sites.

I suspect that Sal I is causing the problem just based on its reputation as a problematic enzyme. The PCR products were designed with 6 flanking bases on either side of the restriction sites (tcgaagTCTAGA... and tcgaagGTCGAC...). This should be enough for digestion but given that the enzymes worked well on the vector i cant see how it could be anything else but poor digestion of the PCR products. NEB says that Sal I digests PCR products poorly but there's no explanation of why. The flanking bases i used on each reverse primer were the same, but could that be the problem? It seems a possibility but there's no mention of site preference for Sal I in the literature (http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/site_preferences.asp). If anyone has any experience with Sal I, any papers or any ideas or suggestions on what to do next that would be greatly appreciated.

Cheers, Rob

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I have just been cursing SalI also........so I'm no help I'm afraid

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I have a couple of suggestions but I've never tried them myself so they may not be that great.
1. NEB make a version of SalI called SalI-HF which cuts iin bufer 4 and is supposed to have reduced activity. You could try using this instead. This also leads to the possibility that you are seeing star activity and hence cleaving either your vector or insert at a sie which does not leave compatible ends. How much enzyme do you use and how long do you digest for?

2. Use a different enzyme. I assume you have already thought of using another cloning site and can't so either you could use XbaI on both ends and screen for orientation or use a site that is compatible with SalI (assuming this doesn't cut in your insert) like XhoI, although XhoI can be a pain sometimes as well....

Hope this helps.

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Hi there,

What about subcloning your pcr product first into a cloning vector like pGem T-easy and then cutting it with the enzymes you added to your primers? should show you, if the digest works (because you cut out your fragment which has a know size and is well visible on the gel) and in addition overcome the problem of limited digestion of the pcr product. As an alternative you could maybe replace the SalI site in your primer with XhoI (compatible ends) which cuts better than SalI.

Stardust

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Ah yes, i was going to ask about that, i was reading about their new HF series yesterday - interesting stuff. They're pretty new so i dont know if anyone knows too much about them but i'll probably give them a try at some stage and will definitely try SalI-HF if i run out of alternatives here. Their main emphasis seemed to be on reducing star activity but i dont think that's the problem here, i haven't had any signs of anything other than my expected bands. To answer your question, approximately 10U/ug for 1 hr @ 37C, pretty standard.


Unfortunately, yes im limited to Sal I/Xba I or a single site cloning of either. I also have EcoRI in the area and i would use that but it's in all the inserts. I like the idea of Xho I though, that's a beauty (assuming Xho I is not in those inserts). That will also be a beautiful test of whether Sal I digestion of the insert is the problem (not the vector). I love that idea, beautiful test and gives me what i want if it works, i'll definitely do that one.

Cheers for your comments, very useful.

Rob

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Another excellent idea stardust, thanks for your input. Too right, that would be a nice way to avoid PCR digestion. Unfortunately, we don't have pGEM T-easy or the like, so we'd have to buy or import the vector from another one of our sites who may have it. Yes, i think Xho I is the go, i'll look into that first thing next week.

Thanks again,
Rob

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Thanks for your posts guys. I'll keep you posted on how i go once i get the results in. Too much suffering falls at the hands of Sal I and definitive answers and solutions are needed on how to beat this enzyme.

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Just to let you know that XhoI can be a bit funny cutting PCR products - check how many bases overhang NEB recommend. Also, I seem to remember that they quote it as having fairly low activity so you may want to digets for a bit longer (although incomplete digestion would be less of a problem on your insert than on the vector). Good luck!

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Thanks for the advice, i dont think ive ever cloned with Xho I before. I routinely add 6 flanking bases.

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How long have you digested wit SalI? Try longer digestion times. It might not work 100% efficiently but it will work.

I got only a few more colonies in actual ligation vs control but I got my clone when checked them.

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