Dear Researchers,
I have custom designed mammalian vector pcDNA3.1 containing the eSIBR region that has the BIC sequence and GFP downstream to the CMV promoter. The eSIBR region has a polylinker that is 40bp with a miR155 back bone. My aim is to clone the siRNA sequence (64bp) in the place of the polylinker. Therefore, I have designed the siRNA with miR155 backbone (64bp) to replace the polylinker.
1. So, I start with the annealing of oligos at 95 deg C for 5 mins with equimolar concentrations of oligos (2uM) as per the sigma protocol with 1X Annealing buffer and let it cool down by itself to RT for about 45mins-1 hour. Then these oligos are treated with phosphonucleotide kinase (PNK enz and PNK buffer) at 37 deg C and inactivated at 65 deg C. Then this oligos are quantified showing the concentration ranging between 100-150 ng/ul. Based on this, I ligate them into pcDNA3.1-eSIBR-GFP (V:I = 1:3) overnight followed by inactivation of T4DNA ligase at 65 deg C. Thereafter, I follow the universal protocol for E.coli DH5alpha (CC) preparation and transformation.
2. Secondly, I even tried to treat the oligos separately with kinase and then go for annealing with the same protocol. Once I m done with this, I could see the difference in concentration of kinase treated-annealed oligos 4 times higher than the annealed-kinase treated oligos (between 400-500ng/ul). Based on this, I ligate them into pcDNA3.1-eSIBR-GFP (V:I = 1:3) overnight followed by inactivation of T4DNA ligase at 65 deg C. Thereafter, I follow the universal protocol for E.coli DH5alpha (CC) preparation and transformation.
Could anyone help me out in this by your valuable suggestions to overcome this issue? As this is the first step of my work, I m terribly stucked off with this. Any suggestions regarding this are welcomed and thank you in advance :)
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