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tricky digestion for cloning gurus - (Jan/24/2006 )

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hey folks. I have been struggling with a cloning project lately, and I am quite certain that the problem is because the two RE's I'm using in the MCS are very close together, in fact there is one base that overlaps the two sites.

I am using BamHI / XhoI, following a published protocol so I know it can work. here is the vector map for pET-14b: http://www.emdbiosciences.com/docs/NDIS/TB044-000.pdf

I have tried this 4 times now and I am not getting anything that is not re-ligated vector.

well, I have run gels at all conceivable steps and the last time I ran through the protocol I called NEB to get their idea of the best reaction conditions to favor cutting by both enzymes, and I did everything they said, and I still got a bunch of vector on my plates. I know both enzymes are good and I know that my vector is linearizing well from the bands that I see on the gel; this is why I suspect that the problem is due to the proximity of the sites.

Does anyone have any ideas to help me figure out a good way to get both enzymes to digest properly? has anyone experienced such a thing?

Thanks to anyone who can help!

A

-aimikins-

Hey aimikins,
Sorry to hear about your troubles. You say it has been documented that this has worked before? I didn't think overlapping sites would work together since many enzymes need the site to be:

1) double stranded, and
2) have at least 2 bases present after the site in order to cleave.

If the sites are lined up as is stated in the manual it would look like this:


----GGATCCTCGAG----
----CCTAGGAGCTC----

when one enzyme cuts (we'll say BamHI) it would yield:

----G + GATCCTCGAG----
----CCTAG + GAGCTC----

OK I would suggest one of two things, 1 use NdeI.

Or two, try using BstKT I instead of BamH I. It uses the 4 base sequence internal to the BamH I sequence (GATC) and would leave a single base prior to the Xho I site. Unfortunately you will probably find other BstKT I sites since it is only a 4 base cutter sad.gif .

If you do find more, try Taqα I or TthHB8 I instead of Xho I. Also internal 4 base cutters, so check if there are other TCGA sites in your vector.

-Captain_DNA-

Dang, I looked through the sequence and those 4 base cutters are indeed everywhere. Looks like Nde will be your only choice mad.gif .

-Captain_DNA-

I have followed the published strategy to get an in-frame gene for expression. I went through the isoschizomer chart with both vector and insert sequence and was unable to find one that didn't whack my DNA into 60 pieces sad.gif

I have tried to email the author of the paper, but their address bounces back; I have a message into their institution and I'm hoping I can get some new contact info.

I think I will have to go back to the drawing board and design a separate primer for one end, so I can get the insert in-frame with another site. dammit dry.gif

I have tried a couple different sequential digestions; some of the reason this has been so tricky is due to the BamHI...a wonderful cheap enzyme with star activity that can't be heat-inactivated. aarrgghhhh

OH well. I have about given up on the published method.

-aimikins-

Oh, hey, I have nothing against Nde, cept it cuts my insert twice.

I'm wondering how the hell the authors got it to work????

-aimikins-

btw, thanks for your help, Captain DNA!

-aimikins-

Can you send a link to their paper? Maybe we can find a clue there?

-Captain_DNA-

http://www.jstage.jst.go.jp/article/mandi/47/1/81/_pdf


I am cloning the LukF-PV into pET-14b, same S aureus strain for the insert, same vector, primers as published. The only thing I am doing differently, I am using JM109 instead of DH5a for the initial cloning, but that shouldn't matter in the slightest for what I am doing

The details are very sketchy in the paper. I have done all the steps in the routine fashion...gels at various points to make sure the DNA is there and that sort of thing...qiaquick clean-up columns (although the first time I used gel extraction through glass wool; I went to the columns trying to beef up my insert:vector ratio and get a little less sample-loss)...ligation o/n at 16C, the sorts of routine cloning steps that always work. I also cloned S and got that one to work on the first try, but I used a different vector rolleyes.gif

I am wondering if someone screwed up in the methods section and it was a different pET vector after all?

-aimikins-

That is a tough one... I've used Nde I and Bam HI to open pET vectors, but not Xho I and Bam HI.

I suppose you could cut the vector with Bam HI, and clone some irrelevant piece of Bam HI digested DNA into it that has no internal Bam HI or Xho I sites. Then cut those plasmids with Xho I, then with Bam HI, and recover the appropriately sized vector fragment that way.

-HomeBrew-

after much sober reflection, and banging my head repeatedly against the wall, I have decided to go with the BlpI site a little farther down. I will order a new 3' oligo and start over.

I suspect it will be faster in the long run rolleyes.gif

thank you guys for your help!

-aimikins-

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