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RP2358's Content

There have been 4 items by RP2358 (Search limited from 29-May 19)


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#167890 Fluorescence micrograph editing in ImageJ, Inkscape, or Gimp?

Posted by RP2358 on 18 May 2014 - 03:04 PM in General Lab Techniques

Hey guys, so I'm having some trouble editing micrographs for my figures in an upcoming paper.

We have cells with different regions fluorescing different colors, and my collaborator sent me the greyscale images of these cells. I know how to simply merge pseudocolor images by RGB channel, but the problem is the images I was sent aren't perfectly aligned. What I need is the ability to slide the two images over one another so that they are properly aligned. I have done this in the past, but I believe it was with native microscope software I no longer have access to. Do y'all know of a way for me to do this with ImageJ, Inkscape, or Gimp? Or do I need to have my collaborator mess with it with his software?

It's easy for me to adjust the opacity of one image and slide it over the other, but then I have to worry about how the background obscures the image I am merging into. Does this make sense? Thanks for any help.

 

No need to double post - I've deleted your other one.  Bob




#158765 Puzzling difficulty cutting out one gene sequence for another

Posted by RP2358 on 08 August 2013 - 08:42 PM in Molecular Cloning

Also, if my pAB were not being cut, then why is my backbone-only control not producing colonies?




#158764 Puzzling difficulty cutting out one gene sequence for another

Posted by RP2358 on 08 August 2013 - 08:40 PM in Molecular Cloning

I'm sure that pAB is since I can see the proper bands on a gel.  Since I am cutting C with the same enzymes, I have just assumed that it is being cut, though I have not been running it on a gel because I'm being forced to work with low quantities of DNA.




#158762 Puzzling difficulty cutting out one gene sequence for another

Posted by RP2358 on 08 August 2013 - 08:09 PM in Molecular Cloning

Hey guys,

 

I have a multi-domain recombinant protein I'm trying to clone.  So let's say I have plasmid-gene pAB and I want to cut out B and put in C, gaining plasmid pAC.

 

To do this, I have been cutting out B with the restriction enzymes MfeI-HF and PstI-HF, as well as the restriction enzyme BsaBI in order to further cut B in half. I then cut out my backbone and purify it.  As for C, it is a free oligo, so that I have just cut and then purified it without running it on a gel.  The last time I attempted this, I further used antarctic phosphatase on my backbone (pA) to prevent it from closing on itself or reincorporated any of the original insert ( B ) that made it through.  I then ligated my pA with C in a 1:6 molar ratio, inactivated it, and transformed cells.

 

Here's the kicker that is showing up repeatedly: my control plate, with only the backbone, will have 0-3 colonies. My ligation plate will have 100-200. And yet restriction analysis (with HincII and AgeI-HF) vastly shows the damn original insert.

 

What the hell could be going on? I'm about to test my intended insert and the primers I'm using to amplify it to rule out contamination by the unintended insert, but that would be very surprising.

 

Do y'all have any other ideas?

 

Thanks.





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