Restriction enzymes not cutting - (Oct/10/2008 )

I was wondering if anyone could help me.

I am trying to cut a gene out of pcr 4 Topo with two restriction enzymes, NdeI and NotI. I have tried a double digest with the two restriction enzymes but my gene did not digest out.

So then I digested with NdeI and another restriction enzyme in pcr 4 topo and set up a second seperate digest with NotI and another restriction enzyme in pcr4 and both digests gave me a product which suggests to me that the sites are ok.

I have tried adding twice the amount of NotI as opposed to NdeI but to no avail. I have tried cutting with NdeI in its optimum buffer, cleaning it and then cut with NotI in it's optimum buffer but have not got a product. I have used new buffers, BSA and enzymes but still nothing. Can anyone suggest a reason why I now cant cut DNA which I have previously been able to cut with the same restriction enzymes?

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HI ,

Could you tell me more about ur work i.e

1. size of the gene
2. RE located on the gene
3. what "other " restrcition enzyme that you choose
4. what u mean by " have not got a product"

The NdeI site is absent on the pCR4 TOPO but if u were to use EcoRI + NdeI then u surely will get a band...that's kinda like my concern.

What i would suggest is

1. Have a stock of ur plasmid uncut.
2. Digest with NdeI first
3. compare the uncut and "NdeI cut" on gel
4. if no difference = NdeI dead or buffer problem or bad quality plasmid.
5. if got difference i.e plasmid is linearized
6. purify and proceed to cut with NotI
7 Then check with gel again.

here's the NEB recommendation for u
Digest in NEBuffer 3 + BSA at 37°C.
At least one enzyme has < 100% activity ( that would be NdeI) in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.

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Hi,
The size of my gene is 750bp. I ligated the gene inbetween a promoter and a terminator, so the cassette is: promoter, my gene, terminator in pcr4 topo. The NdeI site is at the beginning of my gene and the notI site is at the end of the terminator. I have tried doing what you have suggested and I could not digest the gene and the terminator out.

I sent some of the colonies for sequencing to make sure that they were correct and I have now discovered that the promoter and terminator have a lot of base changes and deletions (which is why I cannot cut with NotI) yet the gene sequence is perfect. I sent the original DNA for sequencing as well i.e. just the promoter and the terminator in pcr4 topo and that sequence is perfect. So my problem seems to be with the ligation.

When I try to ligate the gene into the vector i.e. in between the promotor and the terminator in pcr4, something weird is happening with the vector causing base changes and deletions but the insert remains ok.

For the ligations I,
Digest the insert and the vector,
Use SAP on both the vector and the insert to dephosphorylate,
Gel extract the vector and the insert,
Use T4 ligase and ratios of 1:3, 1:5 vector:insert
Transform into e.coli
Then check for insert by mini prep & restriction enzyme digest.

I have never experienced a problem like this before. I cannot understand why the promoter and the terminator are being affected in this way yet the insert remains perfect.

Any help would be much appreciated as I am at a loss as to what is happening.

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Erm,, u can't SAP both vector and insert and for ur case it's not even needed as you're doing double digest. As long as ur digestion is complete you're fine. It might damage the end and affect ligation outcome.

pcrTopo4 doesn't have an NdeI site on its own so why did u digest it with NdeI?

U should use TA cloning kit in my opinion... PCR the reaction with Taq and just proceed with TA cloning.
Since u have pcrTOPO4 , u should have the kit lying in ur lab as well.

In my opinion there's something going on weird with ur cloning strategy. might want to revise..

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Thanks for your help. I've taken your advice and I have come up with a different cloning strategy.

Thanks again!!!!

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