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I feel like a waste of time, and grant funds


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#1 KhaleesiDany

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Posted 04 May 2015 - 02:25 AM

Greetings people

 

I am a Msc student. I started my first year in 2014, well my project only started in June which means in a months time I would have been working on my project for an entire year. So basically my project entails the generation of mTTR ( a liver specific promoter) driven rTALES for inactivation of Hepatitis B virus. My cloning of this rtale has been going horribly wrong fro the last 6 months, it is so depressing, nothing has been working, I have been trouble shooting an optimizing but nothing. I was on the last two stages of the generation of this rtale when things just started going downhill. A PhD collegue also struggled with this rtale plasmid for a while but she resolved its cloning with heat inactivation of the ligation, however that never solved my problem. My course is only 2 years, after the generation of this rtale plasmid I still need to do transfections, expression assays, toxicity assays and others all which i have never done before so i do not know how long it will take me. I am stressing out big time. At the begining of my masters a student suggested an easier way for me to generate this rtale plasmid without all the complicated cloning steps that my project came with.. his idea was to use gibson assembly. my supervisor said we will consider it if all this fails.. now close to a year down the line my supervisor says we should get part of my sequences made by a company and i would just then cut out and insert it into my desired backbone n we will have the rtale (thats if ligation would work, cos i have been struggling to get the sequences i generated into the same vector i dont really see how a company generating it would help).. I feel like i wasted a whole year for nothing the sequences they will be generating i did it, but i did it the old school way, not like the technology that this company (GENscript) uses. I had to do PCR of two different parts of a plasmid, clone them individually into pTZ, screen for positive clones, sequence, cut out relevant fragments to clone together so i could have this two PCR fragments together, screen, n then try to get them into the backbone but it just wouldnt go in, from there i still needed to insert a third sequence into the plasmid to make the final product.. they going to generate all three sequences in the right order and i just have to cut out once and ligate... why could i not have done this before, how much resources have i used up all for nothing. Also now my project is smaller regarding cloning its just one step what if my project gets dissed as not enough for a masters by the assessors panel?  all this makes me worry My supervisor said it was surprising that the lab manager suggested the easier method becasue he is the type to say if you cant do cloning than you don't belong in this lab, that just broke me into pieces, i feel so dishearten, so useless, i know this was not my supervisors intention but yea that really got to me. I even feel like just giving up because i feel like im still going to struggle to get the newly generated sequence into my back bone, my supervisor says it might be easier because we going to use 2  different enzymes to cut and clone with instead of  the two sites that were generated by the same enzyme. to me that reason is not convincing enough, what if it fails then i wasted money to generate this sequence from Hong Kong. How will they look at me then...another thing is every monday we have lab meetings where two members of the lab present a journal article and the other a lab progress report.. my last progress report was in early march its going to be my turn soon again and what will i show them then i have made no progress, this stresses me out so much,  everyone will be looking at me judging me from their seats i can already feel it, oh shes not worthy to be in this lab...Im sorry rfo bad grammar and sentence construction hope i didnt confuse you 



#2 Michael Starr

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Posted 11 February 2016 - 04:05 PM

Well, it is comforting for me to hear that I am not the only one who has felt this way in a lab before.






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