Rabbit anti mouse hrp conjugated secondary antibody

We obtained plasmids containing GFP-tagged CRISPR-CAS9 and guide RNAs targeting the first protein-coding exon of ALKB2 from Sigma. We used Lipofectamine transfection to introduce this plasmid into mouse ES cells and after recovery for 18 hours at 37°C, we used FACS to sort GFP positive cells into individual wells of a 96 well plate. We screened the resultant clones for ALKB2 using PCR across the targeted exon searching for apparent size shifts. We then used Sanger sequencing of the PCR products to select clones showing indels in both alleles. We confirmed ALKB2 protein reduction using Western blot with anti-ALKBH2, a mouse monoclonal antibody (C-9; Santa Cruz) using a Rabbit anti-mouse HRP-conjugated secondary antibody (Abcam). To test sensitivity to MMS, cells were treated with 200mM MMS for one hour before the MMS was washed out. We then sorted single cells using FACs and counted colonies formed after 5 days, comparing to a control treated with 0mM MMS for each line.