I lost my insert! - (Oct/02/2005 )

Yes Homebrew actually you drive my thoughts towards the E.Cloi strain
that what I found
* Cells contain pREP4 plasmid encoding lac repressor in trans, ensuring tightly regulated expression


So it might be the explanation since you've selected on plates with amp and Kana you might end up with colonies of the pREP4 plasmid alone and no pQE vector !

pesji

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Further info from Qiagen's website (emphasis added):

I'm still thinking competency...

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Good point Homebrew but still if you think about competency how would you explain that he had 17 clones ? If the competency would be bad he would rather have no colonies or very few isn't it ?

Furthermore why competency problems would affect the type of plasmid obtained ? We still don't have an answer about the real pattern of his clones. Not trying to get the last wordbut rather trying to help like you do

I would like to see a restriction digest of the DNA of thes clones cause pCR is sometimes just not working I had this problem one week ago with pET Blue2 recombinant tested by pCR with T7 and Blue down primers ! They were negative by pCR but after doing a miniprep I realize that they were some positive recombinant clones so I wouldn't trust to much pCR !

Pesji

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My dear friends,

Thanks for you guys to trouble shoot for me.
I have al thinking that my problem is on ligation.
Could it be the pQE30 vector gets circularized without insert?

Best regards

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I would add a couple of nit-picky things about the transformation protocol...I don't know if any of these things will really make a difference but they are something to think about

I have read from a number of sources that it is bad to use more than 5ul of your DNA during the reaction; I usually put 5ul / 200 ul competent E coli and it works pretty well, Hadrian added 10ul/100ul...that's 10% of the total volume and maybe something in the DNA buffer/ligation mix interfered with the competency of the cells.

I would think 17 is a pretty low number of colonies, no matter what they are...I would think the transformation needs to be tweaked even if it's not the only problem...90' recovery is also really long and may encourage many of the e coli cells to kick out the plasmid; 45'-60' should be plenty sufficient

also, as far as your controls, I'm not sure what you did but I would recommend:

1. vector only
2. cells only (plate on abx and non-abx plates to make sure your selection is good and nothing about the procedure is killing them)
3. vector in ligation mix with no insert (to get an idea of % of recombinants that are self-ligated)- this is a ligation control that should be followed all the way through, even when you use a kit
where religation shouldn't happen - DNA is quirky stuff

i also agree that you need to straighten out which abx to use, if there is a problem there you will get nothing to work right no matter what else you are doing properly

good luck, Hadrian!

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Very good point aimikins

Right usually ampicillin resistance in plasmid can be very quickly turn on so 45mn is enough for the culture after transformation ! Longer incubation usually (in the text books) leads to doubling of the individual clones.



Of course it might get self ligated even if TA vectors are usually very efficient and produce low background r e-ligation specially the commercial one

pesji

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I don't even bother allowing for phenotypic lag if all I'm looking for is Amp resistance. Plating the competent cells directly to Amp plates works fine.

But I think Hadrian is also selecting for Kan resistance, and that does require an expression period, though I agree 90 minutes may be a bit excessive...

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You're right but maybe transforming normal host cells like JM 109 or Top 10 will give better results no what do you think ? I'm afraid that this M15rep4 which are made for expression of protein will not be the best choice just to generate clones !

I do routinely cloning in pGEM-T vector, sequencing of the pCR products if OK ligate into the expression vector transform JM109 cells select positive clones and only the transform different expressions strains like TunerpLAC or Origami or Rosetta with few selected clones.

Pesji

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Dear friends,
I am so glad that you guys are helping me to troubleshoot my cloning problem,
giving me so much valuable input
I will work on it and give you guys an update soon.

Thank you very much

Best regards

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Dear friends,


The fragment was successfully cloned into the vector by one modification that is: ligation for overnight. Transformation still carried out at 37 0C for 90 min. I select all the colonies that grow on Kan and Amp agar for PCR. About 70% of them are with the insert after PCR.

So, I suspect that the UA linear vector somehow had self ligate after storage for a year. But, there are still some remain in linear form.

Cheers. Thank you very much.

:D

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