BclI digestion

[pjansen]

Hi, is there anyone who has experiences with BclI restriction enzyme?
I have the next promblem: it won't cut my plasmid! I am aware of the special incubation temperature and the methylation blockage. I collected the plasmid from SCS110 cells (Dam-). Sequence analysis validated the right sequence, but still the enzyme won't cut (another plasmid is digested properly, so the enzyme works perfect...). What else can it be?

Please help me!!!!
Thnx

[MADHUKAR]

Hi, BcII RE cuts the plasmid sequence only when the bases are in their original structure i.e. they are not modified(methylation).
your plasmid is from SCS110 cells which cuts only in the presence of dam methylase.

[pjansen]

Hi, BcII RE cuts the plasmid sequence only when the bases are in their original structure i.e. they are not modified(methylation).
your plasmid is from SCS110 cells which cuts only in the presence of dam methylase.

Thanks for the quick reply, but I'm afraid I still don't get it.
What should I do to cut my plasmid?

[MADHUKAR]

first of all, you should treat your plasmid with de-methylating agent and then use BcII RE.

[pjansen]

first of all, you should treat your plasmid with de-methylating agent and then use BcII RE.

but the plasmid is already unmethylated, because of the scs110 cells, right?

[canalon]

but the plasmid is already unmethylated, because of the scs110 cells, right?

Should be. And since your enzyme work. What we can question is:
- Are you isolating the right plasmid? (I presumed you checked size etc, but sometimes we have problems
- Are you using a good protocol? Less than 10% of enzyme in the mix, right buffer (NEB 3), 50ºC incubation.
- Are your Scs110 what they claim to be? No contamination?

All this is quite basic, and you probably checked but beside I don't see what can be a problem. More details of your protocol?

[Dr.Stein]

WTH

[MrMistery]

What, Dr.Stein, don't you undertstand that "basic" stuff? Me neither This is genetic engineer talk... Let you and me talk animals )

[canalon]

What, Dr.Stein, don't you undertstand that "basic" stuff? Me neither This is genetic engineer talk... Let you and me talk animals )

Not engineering, Bacterial cloning 101
Knowing how to use restriction enzyme and so on. You just learn that fast when you work in molecular biology. Hey, all this nice cutting and ligation in your textbook need some work to be performed, and that is our job to learn how to do it.

Explanation: BclI is a restriction enzyme, which do NOT cut DNA that is methylated (by the dam system of normal E. coli). So he use a strain that is deficient for this system to allow his enzyme to be cut. The said enzyme is old by NEB (new England Biolabs) witha buffer in which the restriction must be performed (at 50C, as said on the enzyme specs). The more enzyme you put in the reaction, the faster it goes, BUT if the enzyme solution represents more tha 10% of the reaction final volume the glycerol (protects the enzyme during storage) in the storage buffer inhibits the reaction.

Is it clearer now?

[pjansen]

The size of the plasmid is right (even the sequence, because I sequenced it). The SCS110 cells were obtained commercially, so I expect them to be good (a control plasmid that was tranfered to the SCS110 cells (same batch) were digested properly with BclI.
Now I'm trying to create my plasmid in 'normal' coli cells, than isolate the plasmid and perform a transformation of this plasmid to SCS110 cells. It is probably the last thing I can imagine that could be the cause. (although I couldn't explain why if this works).

[pjansen]

I finally got ik work!!
Only I can't explain it....
I ligated the insert in the vector and tranformed it to a normal E. coli cell (dam+), then isolated the plasmid, select a good one and tranformed it to the Dam- (SCS110) cells. Apparently using normal E. coli cells for the construction of the plasmid was essential, but I am not able to explain this. Although I am very happy now, I hope someone is able to explain this to me.....