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insert with blunt ends + vector with sticky ends? does it work?

insert with blunt ends + vector with sticky ends? does it work? - Molecular Cloning Forum

insert with blunt ends + vector with sticky ends? does it work? - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #1  
Old 06-27-2008, 07:52 PM
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Question insert with blunt ends + vector with sticky ends? does it work?



the vector was cut by restriction enzymes (XmnI and NotI) so i got sticky ends. I forgot to use the restriction enzymes for cutting my insert so it had blunt ends. I made the ligation with the t4 dna ligase and transformed the product into DH5a. My cells grew very well on the agarplates with ampicillin. Is it possible that the ligation worked even though the insert didn't have sticky ends? What did my cells absorb?
Could the vector be ligated without the insert even though it had different sticky ends?
It was the first time that i tryed to clone DNA. I don't understand what there is happen. Please help me.
thanks
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  #2  
Old 07-01-2008, 09:27 PM
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Default Re: insert with blunt ends + vector with sticky ends? does it work?

Hey Trypsin!
As much as we all want some magic to happen :-), sticky ends don't ligate to the blunt ones.
Most likely the colonies that you see are results of self ligation of your vector.
If I were you, I would
- Redo ligation with a cut insert (I know it sucks to go back, but if you want the results, deal with it)
- Set up a control ligation of vector alone without insert
Also, another potential problem here is that your vector was not cut well with XmnI and NotI - that's why you could have high background. But first, redo your ligation and see how many colonies you have on the control plate.
Good luck!
And let me know how it goes
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Old 07-07-2008, 06:32 PM
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Default Re: insert with blunt ends + vector with sticky ends? does it work?

Hi Andriy,
thanks for your suggestion. I didn't make a control ligation. It's a good idea. I'm going to do that.
I isolated the plasmid-DNA and made a pcr. The result showed me there were inserts in my vectors.
I repeated the dna ligation with the insert restricted (sticky ends) and got colonies after the transformation. But the result of the pcr was strange. some colonies had an insert and some others not.
The other thing what i don't understand is the MW of my inserts (in both cases). they should be 0,25 kb and they are 0,5 kb. What the hell is that??? It makes me crazy.
But at first i will try it again with a control ligation.
will let you know what is happen. bye
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Old 07-07-2008, 06:55 PM
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Default Re: insert with blunt ends + vector with sticky ends? does it work?

Hi Andriy,
can you tell me what star activity is?
Why wasn't my vector cut well by XmnI and NotI? Aren't 10 minutes at room temperature enough? What is wrong with the restriction enzymes?
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Old 07-08-2008, 03:29 PM
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Default Re: insert with blunt ends + vector with sticky ends? does it work?

Ok, Trypsin, that's a lot of questions!
Let's break them into two categories:
1. Relevant to the progress of your project
2. Educational

1. Project questions:

> "I isolated the plasmid-DNA and made a pcr. The result showed me there were inserts in my vectors."
- Apparently your PCR is picking up something unspecific. Once you isolated DNA from your colonies, the best way to check it is to cut with XmnI - NotI

> "I repeated the dna ligation with the insert restricted (sticky ends) and got colonies after the transformation. But the result of the pcr was strange. some colonies had an insert and some others not."
- This is what you should normally expect. For a second I got a feeling that you were on the right track. But... how do you know that those colonies that did not show insert presence on PCR had DNA in the first place?

> "The other thing what i don't understand is the MW of my inserts (in both cases). they should be 0,25 kb and they are 0,5 kb. What the hell is that??? It makes me crazy."
- Another indication that your PCR was picking up something unspecific

Here is my summary:
- Cut all the DNA samples that you isolated with XmnI - NotI and run them on an agarose gel. Since you are expecting a small insert, make sure that your gel is at least 1.25%.

2. And the educational questions:
- Rule of thumb: For XmnI, NotI and most other enzymes you have to run a restriction reaction at 37C for 1 hr. If you still see high background level on your control plate, cut for several hours
- Star activity is property of certain restriction enzymes like BamHI. If you add too much or cut too long they would cleave DNA in random places

Let's keep working on this! :-)
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  #6  
Old 07-08-2008, 04:58 PM
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Default Re: insert with blunt ends + vector with sticky ends? does it work?

Trypsin,
I have an even better suggestion for you. You could eliminate 99% of the problems by just following a good cloning protocol.
If you would like to try that, start here:
1.a Cut 5 ul of you vector with XmnI - NotI at 37C for 1 hr
1.b Run it on a gel along with 1 ul of uncut vector
1.c On the same gel run your PCR fragment
1.d Purify cut vector and PCR fragment from gel slices. Elute in 50 ul
At the end of this step you will know exactly how well restriction went and how much DNA you have
2. Cut your insert for 1 hour. Purify it on a GFX column (or whatever is your favorite way). Elute in 50 ul
3. Run another gel with 5 ul of vector and 5 ul of insert to make sure you did not loose the DNA
4.a. Set up a ligation: 14 ul of insert, 3 ul of vector, 2 ul ligase buffer, 1 ul ligase
4.b. Set up a ligation control: 14 ul of water, 3 ul of vector, 2 ul ligase buffer, 1 ul ligase
Incubate ligation tubes at 16C for 1 hour or overnight
Transform

The two agarose gels that you run in the process will provide great checkpoints for troubleshooting if any problems arise
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  #7  
Old 07-08-2008, 06:28 PM
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Cool Re: insert with blunt ends + vector with sticky ends? does it work?

thanks for the tips Andriy those will help everyone
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Old 08-31-2008, 09:11 AM
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Default Re: insert with blunt ends + vector with sticky ends? does it work?

The last cloning was successful!!! I tried very many controls and Purifications. The time of the restriction reaction was too short. The exonuclease activity of my polymerase was a problem which was eliminated by purification of the insert after pcr.

thank you all for your answers

My next problems are posted at the westernblot and recombinant protein forum ;-)
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