My Project Finally Worked!

Good news has finally come through on my project!  When I came back to school I tried, yet again, to clone my SUMO mutant into GFP and m-Cherry.  However this time I actually was able to get it to work!  I am so excited!  The interesting part about all of this is that I did not deviate from my typical cloning technique.  This time was just the magical time that it decided to work apparently, I guess the stars aligned.  It is crazy (and frustrating) how fickle cloning can be!

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Limitations of this Summer

I feel like I am a little late to the game getting my last blog in, but I was hoping I would have more to say about final data collections than I do.  This summer was definitely a frustrating one-where I felt like it was Groundhog Day because I was trying the same experiment over and over again but I could not get it to work.  I know that cloning can be a fickle thing, but I am so frustrated with myself because no matter what adjustments I made it did not work.  Therefore, I was very limited this summer in moving forward/in other directions with my project.  I think that while I continue this project in the fall, I may have to reassess my approach-potentially using different techniques and assaying in different ways than I intended to.

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Cloning Advice?

Over the past few weeks I still have been struggling with successfully cloning my SUMO mutant into a fluorescent protein.  Every time I try I have successful digests that come out nicely on the gel.  Therefore, I think my issue is somewhere in the gel purification/ligation steps.  After the first digest I usually have anywhere between 50-90 ng/mL of DNA for each sample (vector and insert).  However, after the gel purification this drops to anywhere between 5-20 ng/mL.  I’m concerned that I do not have enough DNA going into the ligation for it work.  I have tried some ligations with an excessively high insert:vector ratio and even those have not worked.  Moving forward, I do not think it is an issue with my sub-cloning methods because once I sub-cloned some pure m-Cherry plasmid along with my ligations, to start a midi prep, and the plates with pure m-Cherry grew while the plates with the ligations did not.  I am considering cutting down my elution buffer at the end of the gel purification from 30 ul to 20 ul in order to increase the concentration of DNA going into the ligation.  Other than that, I feel like my only option is to keep trying and hope that eventually everything works.  I know that many people have had trouble with cloning at times, and I was wondering if anyone had any tips/suggestions of what worked for them?

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Finding Your Low

You know at the beginning of “Bridesmaids” when Annie is talking to her mom and her mom says “you know what Annie? I think this is your low, and once you find your low, that means you can only go up.?”  Well that’s how I felt at the beginning of this week.  After trying countless times to clone my SUMO mutant into GFP (and failing) I felt like I must have hit rock bottom and it HAD to get better.  Unfortunately, I was wrong.

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Cloning Fun

Hello! It has taken me a while to get underway with results from what I’ve been working on because so far I’ve just been doing a lot of cloning (which has been giving me some trouble).  I began by designing and ordering SUMO-deficient mutants of TR using GeneArt.  Then, I worked on cloning these mutants into m-Cherry so that I would be able to visualize the mutant once transfected.  Unfortunately, I have been having some trouble sub-cloning successfully and had to try multiple times to clone the mutants into m-Cherry.  I am 90% sure I have successfully cloned my mutants into m-Cherry as of now (from checking on a gel), this confirmation has just been put on pause for a while because of issues with sequencing.  However, I believe that the sequencer will be working again soon and I will be able to 100% confirm my clone.

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