Why are some colonies blue in my T-vector self-ligation experiment?

  • Thread starter nobahar
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In summary, the conversation discusses a transformation experiment using a linearized T-vector and antibiotic agar with blue-white screening. The purpose was to insert the vector into the lacZ gene, but there was confusion when blue colonies appeared instead of the expected white colonies. Possible explanations include self-ligation of the vector or issues with the preparatory steps. It was also mentioned that a blank control was used to rule out contamination, but no information was obtained from it. Further discussion includes how the T-vector was linearized and the process for the blank control. The conclusion is that the issue may be with the preparatory steps and more information is needed on how the vector was linearized.
  • #1
nobahar
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Hello!

I conducted a transformation experiment and plated the bacteria on antibiotic agar with blue-white screening. The plasmid I used was a linearized T-vector. The insert with A overhangs would insert into the lacZ gene. If it is successfully inserted, white colonies should form. However, apparently it's possible to get blue colonies. I don't understand why. Can the T overhangs self-ligate somehow? Or would it be something in the preparatory steps for constructing the vector - say, the prep was not complete and some circularized plasmids remained or the t overhangs were not added successfully? I was also thinking that maybe the bacteria could remove overhangs?
I included a circularized control, is it merely to cover the possibility of contamination? I personally don't think so, I had a 'blank' colony (no insert included but t-vector was), on which nothing grew. Although this doesn't actually tell me much.

Anyone got any ideas.
Many thanks.
 
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  • #2
How did you linearize your T-vector? For your blank control, did you perform the ligation reaction or did you just transform the linearized vector without a ligation step?
 
  • #3
Ygggdrasil said:
How did you linearize your T-vector? For your blank control, did you perform the ligation reaction or did you just transform the linearized vector without a ligation step?

Hi Ygg, thanks for the response. Thats the thing, I was simply given a linearized vector, no explanation, so I don't know the process for it. For the control, I subjeted iot to the ligation as if there was some insert, but I just didn;t include an insert in the mix. It went through all the same processes otherwise. he blue colonies have to be self-ligation, since its the only way for the bacteria to survive and the colonies to be blue, so it must be in the prep step that I din;t do. Can they even self-ligate once the overhangs are on?

Thanks for the response.
 
  • #4
Yes, the vector should not be able to self-ligate once it has the t-overhangs on it. The fact that you see no colonies in your blank is also a good indication that no self-ligation is going on. Blue/white screening is useful for distinguishing re-circularized vectors from plasmids containing inserts, and so the screening seems unnecessary because your vector is designed to prevent self-ligation.
 
  • #5
Thanks Ygg, it must be in the prep step then. I haven't been taught how that works so I wasn;t aware of the procedure. Thanks again!
 

1. What is a T-vector self-ligation?

A T-vector self-ligation is a molecular biology technique used to insert a piece of DNA into a plasmid vector. This results in the creation of a circular DNA molecule with the inserted DNA sequence in the middle.

2. How is T-vector self-ligation performed?

T-vector self-ligation involves cutting both the plasmid vector and the DNA insert with restriction enzymes, which create complementary sticky ends. The insert is then ligated into the vector using DNA ligase, resulting in a circular molecule.

3. Why is T-vector self-ligation important?

T-vector self-ligation is important because it allows for the insertion of specific DNA sequences into a plasmid vector, which can then be used for various applications such as gene cloning and protein expression.

4. What are the advantages of using a T-vector for self-ligation?

One advantage of using a T-vector for self-ligation is that it allows for directional cloning, meaning that the insert will only be inserted in one specific orientation into the vector. Additionally, the T-vector contains a T-overhang, which helps prevent self-ligation of the vector without an insert.

5. Are there any limitations to T-vector self-ligation?

One limitation of T-vector self-ligation is that the insert and vector must be cut with compatible restriction enzymes in order for the ligation to be successful. Additionally, larger inserts may have difficulty ligating into the vector due to steric hindrance.

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