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Cloning Problem- colonies only have vector

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koko777
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Topic Started by koko777
on 7/8/2009 6:07 AM   
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I PCR my inserts then digest them and my vector Pet28b with BamHI and XhoI. The digestion of the vector with each enzyme is verified on a gel. The I perform overnight ligation in a 1:5 ratio at about 9C. Ligation screen with one vector and one insert primer shows correct bands. Then I transform into DH5a and the trouble begins. The transformation yields lots of colonies, but no matter how many I screen by colony PCR with a combination of vector and insert primers or insert primers only, none of them have the insert. I've screened almost 70 colonies from each plate and no bands.... I've optimized annealing temp. already. What is going on? It's driving me crazy. Also, my advisor says there's a lot of RNA on the gel for my colony PCR, but I don't know why this would be. Could there just be so much DNA in there that it's running as a big blob near the bottom of the gel? Thanks for your help.


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Posted By qinglongyanyuedao
on 7/8/2009 22:22 PM   
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How did you purify your degitions?

I had similar experience several years ago, and found out that the vector just ligated to my PCR primers which could not be well purified out by PCR/DNA purification kit. If you did the same, you will need to do the gel purification for both your plasmid and PCR product digestions

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parvoman
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Posted By parvoman
on 7/8/2009 17:02 PM   
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My guess is that the PCR product is not cut efficiently at both ends. Most people would do the PCR using a proof-reading taq and then insert the blunt PCR product into an Invitrogen TOPO-Blunt plasmid. From there you can then cut it out using the sites that you have incorporated into the PCR primers (Xho I and Bam HI).

Colony PCR is a pretty good way of screening a lot of clones but I would not recommend doing a PCR screen of your ligation reaction because it is just too heterogeneous. And I would expect most of the products that form in the ligation to NOT give you colonies (eg. vector-vector multimers, since you didn't dephosphorylate the ends of your vector).

Also, isn't the 9 degree ligation quite low? I do 16 degrees and a lot of folks now do it at RT, especially if they have dephosphorylated the vector (which improves your efficiency).

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Last edited Jul 08, 2009, 11:04 AM by parvoman

kjosephs
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Posted By kjosephs
on 7/9/2009 19:02 PM   
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I think the previous responses are all good ideas.  Here are a few more thoughts:

I am sure you have double/triple checked the primer design and obviously the PCR is working, but have you revisited whether you provided a sufficient number of bases as overhang for efficient cutting?  Parvoman's suggestion to go through a ligation independent cloning vector would be a work around if this is a problem.  Any chance you have access to some other validated primers with BamHI and XhoI sites that you could use to generate a control insert to eliminate having to trouble shoot the pcr clean up and ligation?

Could you have the primers re-synthesized? On (very) rare occasions the synthesis goes bad enough that a restriction site is mutated.  I am not saying that this is your problem and the oligo qc is much better these days, but it has happened to me once and two other people over the years and was only discovered when the insert could not be cut from an intermediate TOPO or TA cloning vector which was then sequenced to figure out why.

If you are using a proof reading polymerase and you are not immediately purifying the PCR reaction, the polymerase may be (I know this is a stretch) chewing back into the restriction site.  Admittedly, I have never verified that this is a problem, but it is something I try to avoid with proof-reading enzymes.

 



koko777
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Posted By koko777
on 7/10/2009 8:17 AM   
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I purified my digested inserts with the Qiagen PCR Purification kit. When I ran 5 uL on a gel to estimate the amount of DNA I didn't see any bands that would indicate primers still in it, so I think it worked. I used a proofreading DNA pol (Pfx) to amplfy my inserts, but I purified it right away. I tried to use a regular Taq per my advisor's suggestion (he mentioned that chewing issue as well), but it didn't work (I'm thinking it's probably old b/c it hasn't worked on other stuff either). I'll try to buy a new Taq. I'm trying to avoid buying stuff like a blunt pcr cloning kit for financial reasons, but I may have to. My advisor told me dephosphorylation wouldn't really do any good and would just reduce my number or transformants b/c I'm using 2 restriction enzymes, but I may give it a try. I've done ligations about both 9C and RT with the same results. For the base overhang do you mean the number of bases btw the beginning of the primer and the restriction site or the bases btw the restriction site and my desired insert sequence? If you mean the former then I checked and it's ok. I'll try to find some other primers laying around that have BamHI and XhoI sites. Thanks for all of your responses.



parvoman
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Posted By parvoman
on 7/11/2009 12:40 PM   
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Did you say that you did a PCR clean up AFTER the restriction digests? You said that after the RE digests and Qiagen DNA clean up you didn't see any PCR primers anymore: These should have been removed before the RE digest.

ie.

1. PCR

2. PCR clean up, elute in water

3. If not using a blunt cloning kit: Digest with first RE

4. Purify DNA, re-elute in water

5. Digest with second RE

6. Purify DNA, re-elute in water

7. De-phosphorylate the vector (it prevents vector-vector ligations occuring and thus frees up more vector to ligate with the insert (which, even with the extra clean ups and individual digestions will still not be very efficiently digested - believe me.)

8. Ligate as you did with about a 3-fold molecular excess of insert.

9. Do the control reactions (one without ligase, one without insert and one without vector)

If in the final control you get a ladder with more than two bands then your insert will be ligatable at both ends. Ideally, after an overnight ligation you should no longer be able to see any insert molecules at the monomer level. If you can then it indicates that either the insert is non-optimally digested (which will inevitably be the case) or the T4 ligase is not working at its best. You say that 9 degree temperatures usually work for you so this shouldn't be a problem, although I guess the T4 enzyme won't work as quickly at that temperature.

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Posted By qinglongyanyuedao
on 7/13/2009 22:57 PM   
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I think it worth doing that send whatever you have got for sequencing, that could tell you something.

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varsha
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Posted By varsha
on 7/13/2009 11:37 AM   
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I would do these two steps, along the lines of what Parvoman suggested.

1. Depghosphorylate the vector ends. It would reduce the large number of colonies you are getting right now.

2. Digest the PCR product and gel purify it.

If this does not work, I would TOPO clone the PCR product. You could  then digest the insert with BamH1/XhoI before seetting up the ligation with the vector of your interest.



koko777
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Posted By koko777
on 7/15/2009 8:18 AM   
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I know there shouldn't be any primers in the restriction digest, but someone said it might be a problem. The sequencing was just vector.

I went back and PCR-ed and digested again. I tried to dephosphorylate with antarctic phosphatase after my 2nd digest. I added 5 uL buffer and 1 uL AP to the digest and incubated 15 min. 37C followed by 5 min. at 65C. Then I did a Qiagen PCR Purification to remove RE. I ran 5 uL on a gel to see how much I had and...no band.

Since I already had everything ready to go I just went ahead with the undephosphorylated vector and did a overnight RT ligation, including controls without vector. The controls showed one band each for insert monomers and dimers, but no ladder effect so probably one RE is not digesting insert well, even though vector was digested ok. I ran some of the regular ligations too and got bands for insert monomers, dimers, and a very large band, too large to be vector alone, but maybe some type of multimer.

Per my PI's suggestion I will be ordering primers with different restriction sites.



RLS
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Posted By RLS
on 7/15/2009 8:30 AM   
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 You should be dephosphorylating the vector, not the PCR products. Proceed as Parvoman suggested above.

Secondly, you mentioned where the extra bases go for the PCR primers; these should be at the 5' end of each primer. You can check the New England Biolab's website to figure out what the optimum number of bases to add to the 5' end of your primers so that you can efficiently cut. It really would be easier if you could use a TA or Blunt end cloning kit. These truly make the cloning process much easier.



koko777
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Posted By koko777
on 7/15/2009 8:39 AM   
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I did digest the vector, not the inserts and the # of bases is ok.



RLS
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Posted By RLS
on 7/15/2009 8:42 AM   
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 You need to dephosphorylate the vector after you digest it.



koko777
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Posted By koko777
on 7/15/2009 8:43 AM   
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Yeah, I did.



parvoman
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Posted By parvoman
on 7/16/2009 18:41 PM   
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Strange that there was no DNA after the dephosphorylation and Qiagen clean up. I would probably start with about 5 micrograms of vector plasmid. Cut with each of the REs individually with a clean up after each (I use GeneClean). Check a microlitre (of 30-40) on a minigel then dephosphorylate (I use shrimp alkaline phosphatase) about half of the digested vector - as you did in about 50 microlitre volume, inactivate and again, run 1 microlitre of 30 on a gel.

 

The other problem - as you have pointed out is that one of the REs doesn't seem to be cutting your PCR product properly. I guess you have tested the REs each individually on plasmid DNA to ensure that both work since there is no way of telling whether the one used second to cut the vector plasmid actually worked (from the size on a gel or released fragment, as this is too small to see). If the REs are OK then that just leaves the oligo re-ordering to include a longer bit of sequence to cut off. I hope this works for you.

One final thing though: The Invitrogen kit is relatively expensive but it is not the only TA or blunt cloning kit available. Fermentas do a much cheaper one that works fine. I use my own (home made) competent cells though (as I would with the TOPO kit too as I find the TOP-10 cells to be relatively poor). And, remember that your time doesn't come free. I'd get a kit.

Good luck though.

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sinharakesh
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Posted By sinharakesh
on 7/18/2009 14:02 PM   
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As you have tried a lot from previous suggestion and still u have not succeeded, so its better to change primer and restriction site and enzyme and use fresh restriction enzyme with appropriate buffer. Initially do restriction in triplicate of same sample with double digestion and mixed all at the time of purification by column it will increase your amount and most probably it will be visible in gel than compare it with undigested sequence and proceed further as u r doing.



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