Only Plasmid Bone & gDNA E.Coli??

Until today, I still could not figure out the real problem of my pE-MaSpCL. Before sending it to sequencing, I have triple confirmed its presence from the amplification of NT & CT regions using the GSPs. The amplicons's sequences matched with the transcriptome data I previously obtained. So with high confidence, sequencing was performed but alas, the result was not as I expected. Instead, I was informed the analysis yielded only the plasmid bone and some gDNA E. coli sequences..WTF?? I was really down, depressed, angry and speechless. How could this happen? Is it because of its big size of >10kb? I really do not know. Again, I checked back with the amplifications and still the NT, CT and even the repetitives could be amplified. So tired of doing this...


 

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