Cloning: blunt and sticky - (Aug/04/2008 )

Hi, I am trying to clone a 2kb insert into an 8.5kb UAS-attB Plasmid.

1. I have only 1 common restriction enzyme: Xba1 on the 3' end of the insert that matches a site on the vector.
2. For the 5' end I have to blunt a site on my insert(when I cut it out of the vector it is in) and a EcoRI/BglII site in the UAS vector.
3. I have been having a lot of trouble with this reaction.
4. I cut first cut the insert with an NcoI and the vector with EcoRI/BglII. Then I blunt them with Klenow.
5. Then I gel extract only the vector.
6. I then cut both the insert and vector with XbaI. At this point I do a gel extraction of my insert.
7. Then I ligate them.
8. As a control I use uncut vector, and the vector that I cut with eco/Bgl, blunt and then cut with Xba.
9. I saw as many colonies on the plate with the insert and vector as with the negative control.

I have done various steps to ensure that the vector is cut and runs as a linear band on the gel. I changed the choice of enzyme from EcoRi to BglII for the 5' cut of my vector. I did various controls with the vector eg ligating back after the first cut, ligating back after blunting and then transforming versus transforming the unligated vector.

I have been struggling with this cloning for the last 5 weeks.

Any suggestions?

Thanks

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I'm not sure I fully understand the approach you have taken. If I am right you end up with a vector cut with XbaI and an insert that is only cut at the 3' end. Why would you expect the insert to be incorporated into the vector. If I understand you correctly, what you are doing cannot work. All you will get is religated vector without the insert as the insert will be blunt at one end and have a XbaI overhang at the other end so it can't be inserted into a linearised vector which has two XbaI overhangs.

If I was doing this cloning I would use PCR to amplify the insert. For a 2kb insert use an enzyme which is highly processive and has good proof reading activity - Vent (NEB) or Pfu turbo (Stratagene) would be ok. Avoid using Taq in this case.

What restriction sites are available in the UAS-att vector?
You will also need to check if these restriction sites are found within the DNA you wish to insert. I've attached a general cloning guide (Word document) I wrote for an undergrad student which will explain how to design PCR primers, choosing restriction sites etc.
The instructions guide you through the cloning process with an example so hopefully it is reasonably straight forward to understand.

Your plasmid is relatively large so when you get to the ligation step you will want to have 200 ng of double digested UAS-att DNA and about 200ng of the 2kb insert DNA. It can help to heat the ligation reaction (BEFORE adding the ligase) for 2 min at 50oC and then cool it on ice for 2 min. Then add the ligase and incubate at room temp for 1-2 hours. The reason for doing this is that the insert molecules once you have cut the PCR product with restriction enzymes is able to bind to itself to form chains of insert that will then be ligated but heating and cooling them in the presence of the cut vector should promote binding of single insert molecules to the vector and the ligase with complete the process.

Hope that helps.

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I would also use PCR to recover my insert, and engineer appropriate restriction sites into the 5' end of the primers to facilitate cloning into your UAS-attB vector.

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Thanks. For more clarity:

1. I am cutting the insert(from the vector it is in) with NcoI on the 5'end, blunting this end and then cutting the 3'end with XbaI becoz the pUAS-aatB vector doesn't have compatible sites.

2. Then I cut the pUAS vector at one end with either EcoRI or BglII, blunt this end, then cut at another point with XbaI for sticky overhang. I loose about 40 bases in the pUAS vector once both the enzymes cut. Hence the 2 sites in each for cloning. The pUAS vector is about 8.4kb. I am wondering if the size or quality of vector I have might have something to do with cloning inhibition. I was using the vector from a neighbouring lab. I have now generated more on my own: I am repeating the whole thing with newly synthesized vector.

3. I thought of PCR as well, but seems to me that cloning is the issue. Generating the insert from it's vector has so far seemed quite good, becoz I am able to gel purify the insert of the right size once I have cut it out from it's current vector.

I would appreciate of you could shed more light.

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I would agree with HomeBrew, this is an overly complicated ligation strategy and thus as a rule of the thumb prone to failure. I would prefer to PCR the insert with the appropriate restriction sites added to the primers.

However the strategy does seem workable and should work.

Just checking, are filling in the 5' overhangs of NcoI cut insert and EcoRI or BglII cut vector by full fill in with dNTP using klenow, right?

If you are worried that the ligation isn't working, run out some of the ligated DNA (after ligation) on gel in a narrow well. YOu should be able to see multiple high molecular weight bands if the ligation reaction has been successful.

Also note that the T4 ligase buffer is prone to becoming inactivated. The ligase buffer is also the same, the dATP does degrade.

Also note that XbaI is can inhibited dam methylation by overlap. So check your XbaI site to see if they could be inhibited by dam.

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Thanks again to all. I checked my ligase and ligation buffer before. I cut my vector with EcoRI and then ligated with the ligase and buffer in our freezer vs a ligase and buffer from a teaching lab in our institute that has a continous supply of stuff. The efficiencies were comparable. I will try the ligation mix on the gel as you suggest.

Also I will try homebrew's suggestion of heating the mix before ligating. I guess the idea is to prevent concatamer formation. Howver, homebrew, wouldn't using equal amounts of vector and insert pose a problem here? I read in literature that the vector to insert ratio should be 1:3?

Thanks

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Sorry, I guess I will try leonardp's suggestion of heating the ligation mix. Thanks for the word doc! And if i go on to design primers, I agree with homebrew that I must incorporate a restriction site in the 5' region of the insert compatible with a site in the vector.

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No, not really. Vector: insert ratio of 1:3 is normally quoted. But it doesn't do much and I believe it actually makes the ligation more difficult. A high insert ratio increases the likelihood of multiple inserts ligating to the vector... which makes closure of the vector impossible (especially if the insert has dissimilar overhangs.) Draw it out on paper and you will see what I mean

A ratio of 1:1 is work well and works well even on multiway ligations.

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I guess until now the bigger problem is that I only see vector religation even though during the prcedure of cutting the insert, it looks really clean on gels. I see that again: i checked the colonies again and I see only bands suggesting vector religation but not the insert. I am really pulling my hair apart now: my boss thinks I can't do molecular biology!

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A few questions:

Are you sure your double digest is working? Do the sites overlap? How are you sure both enzmyes are cutting? Why do you need to use two enzymes at all, since the next step is to blunt both the vector and the insert?



How is the Klenow removed from the insert + XbaI reaction?

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