| | 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
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! :-)