Dear Researchers,
I have been trying to clone my siRNA which has miR155 backbone into modified pcDNA3.1. I start with annealing of oligos then restriction of vector (1ug/ ul vector conc; 20ul total volume of the reaction), ligation (1:3 vector-oligo, 16 deg C overnight) and transformation. I don't find any troubles with the controls (Competent cells, undigested vector as negative control, restricted vector control and a check plate) I have used. I couldn't get positive clones in my target genes even after many trials. I will be glad to have solutions from any experts.
Thank you in advance
I don't quite follow. Are you annealing the oligos, then digesting them with a restriction enzyme in order to ligate them to a similarly digested vector? In that case, you have to ensure that when designing the oligos, you left a long enough "stuffer" between the restriction site and the end of the oligo, as most REs won't cut efficiently -or not at all- if their recognition site is placed flush at the end of a double stranded DNA fragment.
Another thing, are you sure you ligase and/or your ligase buffer are OK?
In my experience, cloning annealed oligos into a normal vector can get surprisingly tricky at times. I have never been able to find the cause of this problem, and have simply resorted to screening a large number of colonies.
Dear Alejandro,
Thank you for your response, I actually don't restrict Oligos. I ligate them directly into digested vector. and yes my ligase buffer and ligase enzyme are alright!!
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