Ok, scientific community, I come to you in desperation far later than I should have. I can’t clone a cohort of fusion protein DNAs that I absolutely must. The screwed up part: it’s basic cloning. I’m no novice to this, but I feel like one at this point. Here’s what I’ve got:
Insert and vector both double digested with XbaI and SpeI (yes, I know they’re compatible).
I’ve treated the cut vector with alkaline phosphatase and column-purified it.
I’ve tried 2 different AP treatments, 2 different ligases, and 2 different types of competent cells, and fresh plates every time.
All of this leads to one reproducible result: blank plates. Positive control plates flourish, but I might as well be plating Clorox on the experimental plates. Can anyone suggest something? My job may depend on it 
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ive been having similar troubles with my subcloning and after a couple of months one of the protocols i was trying worked
1) shortening AP treatment
2) digesting with more enzyme
3) ligating with more DNA at incredibly high ratios
this helped with a couple of my constructs in pProEX but still not working for my pFastbac .. so i might try my phosphatase treatment for an even shorter time
there are some good ideas here and im glad i ran into this blog entry
also, i use TOP10 competent cells from invitrogen, which i believe are super sensitive to mechanical lysis, so i think ill try shaking at slower speeds even though i was doing it at the standard 225-250 rpm
expression of my insert is toxic for the bacterial cells but i had never thought of it as potentially killing my competent cells. i dont know if that is the problem coz i use SOC medium which has some glucose in it…
Hi all..I am new to this blug.. have gone through it lots of time its great one you guys are doin gr8 job.
I am ligating BamHI cut vector pLAFR3 (SAP phosphorylated) with SAU3AI cut DNA (~20kb) and transformation in HB101, but getting no clonies. I have tried different vector insert ratio but nothing is working.
Wts wrong goin on.. got no idea..
All of your help and comment would be highly appriciated.
Thanks
If it’s a toxicity issue, you might try growing at 30 degC instead of 37. That’s been critical with a couple constructs in our lab that our toxic.
Wow, I really appreciate all of your help. As it turns out, I’m pretty sure I just had crappy AP. Correction, way too good AP. I think Sven got it right on the money with it just not being killed. I’ve got 4 of my 5 clones now, and the fifth is underway.
And pkrug, next time I’ll try the sacrificial doll to the Gods of Molecular Biology. That used to work with the PCR Gods.
I am assuming that this is not an insert specific problem. There are plenty of problems that can come from inserts causing toxicity of various kinds. The simplest cause of your problem may be the AP – if you are using the calf and not the shrimp, it is very difficult to kill. Not killing it can dephosphorylate the insert during ligation – giving you no ligation and no colonies. My recommendation is to do a positive control ligation where an insert has to go in – maybe an incompatible end ligation so you don’t have to use AP. It is best to troubleshoot one thing at a time to locate the source of the problem. Also be aware that blue/white screening doesn’t always work. For short sequences, you often get blue colonies that have plasmids with inserts. Further, blue white screening (induction with IPTG) could mean that your fusion is being expressed in the colonies with the inserts. If the protein is toxic, the colonies with your insert will die and you will never see them. If this is the case, you can supress lac with glucose to drive down expression of the fusion protein and check for inserts using colony PCR.
Good Luck – Sven
Is that CaCl2 transformation? ‘Cause electroporation is usually more efficient. Also if you’re selecting on Amp, you can omit shaking completely and just plate them outstraight away
You could also try one of those strains which supposedly are better when it comes to toxic proteins, but I never worked with them so can’t really recommend anything.
Sorry – didn’t see the “flourish” comment in your initial post. Well, if you are getting colonies with the vector alone, it’s not the shaking. I had been shaking in a vortexing 37°C heat block at full speed for 30 min. Bacteria tend not to like that.
With AP you get zero colonies? I always get a few… If i had to guess I’d say its not the cells or the ligation but the AP rxn. But really zero white colonies with no AP? Maybe the insert is messed up? Are you inactivating the ligation prior to transformation? Maybe something inhibitory in the insert? I feel your pain. Perhaps an offering to the gods of molecular biology is in order? We used to put up a cloning idol made of kimwipes and microcentrifuge tubes over our benches when this sort of thing would happen. (We’re virologists, not bacteria folk, so superstitions abound).
Perhaps migg is right on the toxicity – is the promoter able to be shut off on the expression vector by chance? Can you ligate the insert into some other vector that is not for bacterial expression just to see what’s going on?
migg! not even 20 min in a 37°C water bath or heat block?? oh the poor bacteria! or is that recommended shaking hour just to help pad the manufacturer’s cfu/µg?
I am assuming that this is not an insert specific problem. There are plenty of problems that can come from particular inserts causing toxicity of various kinds. The simplest answer is the AP – if you are using the calf and not the shrimp, it is very difficult to kill. Not killing it can dephosphorylate the insert during ligation – giving you no ligation and no colonies. My recommendation is to do a positive control ligation where an insert has to go in – maybe an incompatible end ligation so you don’t haver to use AP. Troubleshoot one thing at a time to locate the source of the problem. Also be aware that blue/white screening doesn’t always work. For short sequences, you often get blue colonies with inserts. Further, blue white screening (induction with IPTG) probably means your fusion is being expressed in the colonies with the inserts. If the protein is toxic, the colonies with your insert will die and you will never see them. If this is the case, you can supress lac with glucose to drive down expression of the fusion protein and check for inserts using colony PCR.
Good luck
Sven
A couple of questions – are you using the competent cells that you are going to use for expression of the protein (i.e. NovaBlue DE3 or something similar)? Because those cells are dramatically less competent than something like JM109 or DH5alpha. Also, I ran into trouble where I was shaking way too hard after adding the SOC, killing most of the bacteria in the process. So make sure you shake pretty slowly. Another idea might be the AP – I routinely see both loss of vector and some degradation if the AP reaction goes too long or is at the wrong temp.
Hope this helps – p.s. If i recall, XbaI is blocked by dam methylation… but that wouldn’t cause your problems. Have you tried a no insert control to make sure the vector is happy? What about trying it without the AP and just do a brute force screen?
hey pkrug,
I very much appreciate your input. I’ve actually used two different kinds of DH5-alpha from Invitrogen. I’m not trying to get protein expression in the bugs (although, if it is being expressed, there’s a good chance it could be toxic, which could be the problem). I tried it w/o the AP for a while and was getting the opposite problem, ie: overwhelming growth of blue colonies (oh yeah, that’s another thing that should add to the “no-brainer” part: blue/white screening). See, like I said XbaI and SpeI are 100% compatible–something I didn’t realize until too late. They both leave CTAG/GATC overhangs, so the vector will ligate back up in a wink w/o AP. The brute force screen would be swell if not for the fact that all the colonies are blue…
I will, however try the shaking more gently thing. I usually go for 225-250 for an hour. And I also realized that I had not tried the different AP yet. Also, I can try different cells (not from Invitrogen). Thank you very much for your suggestions.
Oh yeah, and I’ve done several no-insert controls, AP and non. Non gives plenty, AP gives none.
Hi, I’ve been having similar problems with some cloning, and I have a question about your final result. If you were column purifying the vector after the AP treatment, how could the AP be affecting the subsequent ligation? Shouldn’t it have been removed in the purification?
Thanks, Dan
Even i faced the same problem for more than six months with the enzymes NdeI and Bam HI…So what u do is just change the vector and insert ratio….Try out 1:6 or 1:8 i am sutre this will work out….
Try good luck!!!!!!!
Nidhi
From India