For all these 10 months, I repeated the same work again and again and again and again. I feel very tired and losing hope. I am almost giving up and I want to stop doing my work.
During the fasting month, after I went back to my hometown, I got an instinct to check again the DNA sequence for the gene I work with. But at that time, since it was a holiday, I decided to rest for a while and helped my mother cooking and everything. I just wanted to enjoy the 'eid.
Then I came back to my campus and I started doing the same work, AGAIN. Today, this morning I checked again the DNA sequence. This time I checked it carefully and tried to not to make any mistakes.
Suddenly, I found out that there are mutations inside the DNA sequence that I work with for 10 months. There are 87 additional nucleotides inside the gene, which I don't even where they come from. When I translated the DNA sequence into amino acid sequence, there is a STOP codon in the middle of gene.
The mutation occurs in the middle of the sequence. The '---' line is the mutation.
Additional STOP codon is highlighted in red box. Because of this codon, the protein did not express.O Allah, Subhanallah, I was like stunning and then I laughed out loud. No wonder I got nothing for the Western blot analysis and protein purification.
Luckily I have 2 more clones that I never used but I kept the glycerol stock. I re-checked the DNA sequence of both clones, I found out that both of them got a good sequence. Perfect alignment and no mutations. Again, Subhanallah...Why didn't I check the sequence before, I don't know.
2 comments:
you are lucky sbb tak payah nak buat site directed mutagenesis sbb ada ex stock.
Ps, nak buat molecular mmg kena lab tak ada gangguan...satu nuclotide silap pun kena check :)
Go go norly
Eh, site directed mutagenesis tu budak FYP kena buat and aku kena supervise diorg. Still dlm kerja aku jugak sebab diorang guna clone aku.
Ha, now u know nape aku suka lab sunyi2. Senang nak fokus.
Post a Comment