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Recombinant construct looks smaller than empty vector


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#1 cerqueiragm

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Posted 24 August 2011 - 03:46 AM

<<<<<<Problems with the characterization of my recombinant clones>>>>>>

Guys, I am a bit frustrated since I don't understand what is going on with my ligations, or minipreps, or E. coli strains, or whatever...

The situation is:

1. Firstly I ligated a 1.2 kb fragment into pCR2.1 (TA cloning). I confirmed the cloning by PCR and propagated the clone for miniprep

2. After the miniprep I characterized the clone again by PCR and Restrict analys., and cut the construct with two different enzymes to insert now fragment two (1.3 kb).

3. I cloned fragment 2 (1.3 kb), heat shocked Top10 cells and colony PCR to identify one recombinant clone that now contains the cassette (Fragmts 1 and 2 fused).

4. I propagated the clone, miniprep it and runned on the gel.

5. on the gel...the recombinant vector containing fragts 1 and 2 looks larger than pCR2.1 alone, but smaller than the recombinant vector containing fragt 1 (weired).

6. In addition, the recombinant vector containing fragts 1 and 2 is resistant to restriction analyses with different enzymes.

7. It is also resistant to PCR amplification using M13 or other primers.

I read that sometimes it happens when cloning large fragments or due to problems with the alkaline lysis (during the miniprep), which leads to an erroneous fold of the plasmid DNA, rendering a molecule that is not possible to be characterized.

This is not an isolated situation, since it also happened to another construction that I've been trying... involving a different fragment and different vector.

I read that re-transformation of this misfolded (or denatured) molecules into E. coli (top10 or DH5alfa) restores the functionality of the molecule.

Before to go ahead and try something I would like to ask for your opinion...have you ever seen a similar behaviour for a plasmid vector? What could be causing such phenomenon to different constructs? pH? Salt? bad luck?

Thanks a lot.
Kind regards.

#2 phage434

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Posted 24 August 2011 - 04:08 AM

I might suggest that it is a bad prep of your plasmid. Inhibitors could prevent RE digestion and PCR, and could affect the position of a band in a gel. If you still have the plate from your original transformation putting the two clones together, I'd go back to that and see if there were another colony. I take it your colony PCR results showed correct band length. I've never heard of un-cuttable plasmid or of using re-transformation to cure it. I suppose a miniprep could be bad enough to make things look like that. How are your minipreps done?

#3 prodes

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Posted 25 August 2011 - 06:35 AM

Hi,

Possible explanation to 5:
The fact that you get different sizes for the vectors with the 1 & 2 fragment and the 1 fragment indicates that you have got different prep and supposedly the larger vector is in a more compact state i.e. it is in a nice supercoil confirmation while the smaller vector might be relaxed (this all depends on the plasmid extraction procedure). That is not surprising if you run uncut plasmids.

Possible explanation to 6:
It is possible to obtain this result because indeed there are enzymes that exhibit different efficiency on different conformation states of the DNA . Therefore, as we elaborated above your plasmids are most probably in two different conformations i.e. pCR2.1 + fragment 1 is relaxed and pCR2.1. + fragment 1 & 2 is supercoiled. Which enzymes are you using?

Finally, I do not have any possible explanation why your pCR2.1 + fragment 1 & 2 would not work for the PCR. Given you have sufficient time at 95degC for the denaturation step there should not be a problem and it should behave the same as the other plasmid. Maybe you could just make sure you have enough time at 95degC. If you are doing colony PCR I recommend initial 5 mins at 95degC and then 2 mins for each cycle. For a regular PCR from vector 2 - 3 mins at 95degC should be enough.

Cheers.

#4 cerqueiragm

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Posted 30 August 2011 - 04:34 AM

Thanks a lot for the comments guys. I decided don't waste more time on crap constructs and started all over during the weekend. Now the new constructs look beautiful and everything is working well (REs, PCR, preps electrophoresis). Certainly I just screwed up at some point!
Cheers.




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