...favorite vector has a relatively limited MCS (BamHI - EcoRI - SalI) and you want to expand that with the addition...add NdeI, PacI, AscI and MfeI sites between the EcoRI and SalI sites of the vector, we design a top oligo...overhangs generated when digesting the vector with EcoRI and SalI (see diagram ). To do this, we add 5' ...of the other oligo), but this would destroy the EcoRI and SalI sites in the final vector. Order the following...conduct a restriction digest of 1μg of vector with EcoRI and SalI. Run an agarose gel and cut out the band...

... digest with EcoRI and NcoI: 1 μg miniprep DNA 2 μL 10x NEB buffer for EcoRI 0.8 μL EcoRI 0.8 μL NcoI ...digestion with AgeI and EcoRI. shRNA oligos are cloned into the AgeI and EcoRI sites in place of the stuffer...with EcoRI. Mix: 30 μL pLKO.1 TRC-cloning vector digested with AgeI 5 μL 10x NEB buffer for EcoRI 1 μL...cases (depending on the target sequence), while the EcoRI site is preserved. For a complete map of pLKO.1 ...1.9kb stuffer that is released upon digestion with EcoRI and AgeI. The oligos from section B contain the ...sequences that are compatible with the sticky ends of EcoRI and AgeI. Forward and reverse oligos are annealed...catalog # AgeI New England Biolabs (NEB) #R0552S EcoRI NEB #R0101S NEB buffer 1 NEB #B7001S NEB buffer ...

...in the following cartoon, in which we are adding EcoRI and NotI sites to Your Gene of Interest (YGOI) for...will save time later. In our example, we will use EcoRI and NotI to ligate our cDNA into the recipient plasmid...the region that binds the ORF and we will add the EcoRI restriction site (GAATTC) to the 5’ end of this ...

...ligation of two sticky ends that were generated by EcoRI digestion: Usually, scientists select two different...