Block-it Plasmid Ligation - (Sep/07/2006 )

Hi there!

I just wondered if someone can help me... I bought the Block-It RNAi kit from Invitrogen and they sell the plasmid in linearized form. I would like to make more of the plasmid but i can't find out with which enzyme they are cutting the plasmid. The map looks like that (doublestrand):

backbone-AAGGCTGTA CAGGACACAAG-backbone
backbone-TTCCGACATACGA TGTGTTC-backbone

so there are a ACGA overhang and a CAGG overhang. I can't find out what to do to make more plasmid because I can't find enzymes which leave those overhangs...

Any Idea?

Thanks Stardust

design oligos so that u have some site which will be located a few bases from the overhang but digest it such that u have the proper ends.

like an enzyme which will leave a 3' overhang and cut a few bases from the actual site.

Stardust, the enzyme finder on website of NEB and Fermentas is a good first port of call, to design the modifying primer. On the NEB website I was not able to find for certain an enzyme that gives the AGCA 5' overhang. However I was able to narrow it down to BsmFI, BtgZI and FokI. There was not enough sequence information supplied in your post to accept or eliminate these three candidate. You will have to check your plasmid and see. I have also not looked at Fermentas.

The 5'CAGG overhang on the otherhand is a snap. there are lots of enzyme that will produce that overhang, you just have to engineer the right binding site 5' upsteam to that sequence.

If you are willing or allowed to modify the few bp directly upstream of the AGCA overhang, then it would be even easier to find an enzyme.

Good luck.

I think "BsmBI" from NEB might work for u.

It will cut a few bases after the site so u could design something in between and have some spacer between the 2 sites.

Thanks for your answers...

BsmBI seems to be what i need for the second overhang! Thanks!

But i still can't find a way to generate the ACGA overhang...this is really difficult for me...

Any ideas? I checked Rebase, NEB, Fermentas but can't find what i need...are there enzymes that recognise a sequence and cut in front of it??

Thnaks Stardust

Arg... brain has failed me! Do I look silly.

Scolix is correct. And I was spouting quite a bit of rubbish.
As Scolix suggested, have two type IIa restriction enzymes like BsmBI placed divergently from each other. Put in the right base pairs and you'll have your ends.

As it is easier to see understand a drawing... (and as penance for my stupidity)... please look at the illustration below



I have used BsaI from NEB in my example, as the enzyme is about 6 times cheaper compared to BsmBI. The recognition sequence of BsaI is GGTCTC N | NNNN

The run of A s is just a spacer, replace it with any suitable nucleotides.

The first BsaI is on the complementary strand, facing from right to left... using the right base pairs you'll get the ACGA overhang. The second BsaI site on the forward strand, with the right base pairs give the CAGG overhang.

I hope this clarifies Scolix's explanation.

Thanks perneseblue for clarifying my point.

That looks perfect!!!

Thank you so much!!

Stardust