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

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Dear Forumers,

I have one problem with my cloning. I am trying to clone an outer membrane protein (OMP) gene (from bacteria) in to a plasmid pQE 30 (Qiagen Ua cloning kit). After ligation of my PCR product with UA-pQE30, i transfect the plasmid into my competant cell (E. coli M 15 strain) and growth on ampiciline plate. I got 17 colonies at the next day.
I thought my cloning was successful. However when I pick those colony and do a PCR on it, I got no band. I lost my insert but somehow the the UA vactor seem to be ligated itself and successfully enter the competant cell. Why? How?

Any solution will be greatly appreciated. 10,000 thanks for helping me.


Best regards

-Hadrian-

hadrian, what are the details?

was there a problem where the ligation occurred? have you linearized the plasmid you obtained, or better yet sequenced it, to look and see if somehow during the ligation you lost part of your intended insert, and your primers no longer recognize their template? perhaps your insert is a truncated version of what you were attempting to get?

-aimikins-

Could this just be a failed PCR? Did you run a positive control?

-HomeBrew-

Dear freinds,

Is there any way to know weather my ligation is done properly or not?
I do not linerlised my plasmid yet. I am going to do so.
I am also will PCR it using sequencing primer provided by the kit (flanking the cloning site).
I don't think I my PCR problem because my +ve control turn out positive.

However, when I done all this and yet fail to find my insert, what do
you guy recomend me to do?

Thank you very much.


Best regards

[

-Hadrian-

It is not unusual for such a cloning to produce some vector-only transformants. I use the Invitrogen TA cloning kit (which offers blue/white selection), and there are always some blue (no insert) colonies. These are usually vastly outnumbered by hundreds of white colonies, however...

Can you give us details about how you proceeded:

  • How successful was your original PCR, from which your template came?
  • Did you gel purify your PCR product before attempting to clone it?
  • What type of polymerase did you use to generate the template?
  • Did the competent M15 strain come from Qiagen or did you make the strain competent yourself?
  • If the latter, what procedure did you use?

-HomeBrew-

Getting false recombinant colonies might just be due to residual supercoiled vector carried along during vector preparation:

Supercoiled DNA give 100 times more colonies than a cohesive ligation

Second remark I usually transform recipient E.Coli which don't express the protein like JM109 or Top10F and when I get the correct clone (insert in and sequenced) Then I just transform the expressing strain (in your case M15rep) with the SC DNA from my clone.

pesji cool.gif

-pesji-

Dear Homebrew,
1) I think my original PCR is successful, as I can see anice 1215 bp band.
2) I did not see any primer dimer on my gel and no unspecific band, thus I did not gel purify my
band. I just do a simple PCR clearn up.
3) I am using BioTherm Taq, GeneCraft. I am not sure weather this Taq will produce a 3' overhang
A (essential for UA cloning) or not. So I do both type of ligation with and without A addition.
4) Mother cell of M15 strain was provided by Qaigen. However it is not enough for my whole project,
so I growth them in media with kanamycine.
5) I incubate my PCR product together with my UA cloning vector in 2x ligation mix (provided by kit)
for 2 hours. PCR product : vector molar ratio = 5:1.
Then the 10ul of ligation mix was mix with 100ul of competant cell (M15 strain E. coli), Keep on
ice for 20 min and heat it uo to 42oC for 90 sec (water bath). add 500ul of Psi brothand
incubate for 90 min at 37oC. Then Plate out 50, 100 and 200 ul of culture on agar plate
containing 25ul/ml kanamycin and 100ul/ml ampicillin. Incubate the plate at 37oC overnight.


Thank you very much.

-Hadrian-

QUOTE (pesji @ Oct 4 2005, 06:43 AM)
Getting false recombinant colonies might just be due to residual supercoiled vector carried along during vector preparation:


Dear Pesji,
What do you mean by residual vector carried along during vector preperation?
Are you suspecting that some other plasmid had enter my competant cell instate of the actual plasmid?

Thank you very much for your input

Best regards

-Hadrian-

QUOTE (Hadrian @ Oct 5 2005, 06:01 AM)
4) Mother cell of M15 strain was provided by Qaigen. However it is not enough for my whole project, so I growth them in media with kanamycine.


Okay, I doubt there's a problem with your original PCR. The Taq should be fine (is this it?) and should leave TA overhangs. I would've gel purified my product in an agarose gel with guanosine as a UV protectant, but I don't think this is your problem.

I guess my biggest concern is your homemade competent cells. After you grew up some more M15, by what prodedure did you make them competent? Did you try transforming them with some other plasmid DNA to see if they were competent?

-HomeBrew-

QUOTE (Hadrian @ Oct 5 2005, 11:05 AM)
QUOTE (pesji @ Oct 4 2005, 06:43 AM)

Getting false recombinant colonies might just be due to residual supercoiled vector carried along during vector preparation:


Dear Pesji,
What do you mean by residual vector carried along during vector preperation?
Are you suspecting that some other plasmid had enter my competant cell instate of the actual plasmid?

Thank you very much for your input

Best regards

Sorry I assume that you've prepared yourself the vector but reading more carefully it looks like you use a commercial vector with TA cloning so my explanation doesn't work in this case.

Well that looks strange cause I routinely use the TA vector either pGEM-T or pCR Topo and bnever have this trouble blink.gif

Well after long reading in the Quiagen website I think I might have found a possible problem in your procedure

The vector pQE contains
beta-lactamase gene (bla) Confers ampicillin resistance

they don't mention any Kanamycin resistance so if you select with Kanamycin you might run into trouble !

Did you try to cut your minipreps with BglI wich is cutting into the bla gene from the vector at least it would indicate that you're dealing with the right plasmid !

I know it's tough but we will try to give as much help as possible !

pesji cool.gif

-pesji-

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