Pancreatic alpha Cells

Transformation of Max Efficiency DH5α competent cells was modified from the manufacturer’s protocol as follows. 25 μl of cells were used per transformation, corresponding to one fourth of the recommended cell volume. Cells were transferred to 2 ml polypropylene tubes (Axygen, Union City, CA). DNA was diluted and mixed in Milli-Q purified sterile water and 2.5 μl was added per transformation. No difference in transformation efficiency was observed when the DNA was prepared in 10 mM Tris-HCl buffer, pH 8.5, with or without 1 mM EDTA. Cells and DNA were incubated on ice for 30 minutes and then placed in a 42°C water bath for 45 seconds. Following a two- to five-minute incubation on ice, 225 μl of room temperature SOC medium (Life Technologies) was added to the tubes, and the cells were allowed to recover at 37°C with shaking at 250 rpm for one hour. Cells were then plated on LB-agar plates with appropriate antibiotics (100 μg/ml ampicillin, 60μg/ml kanamycin) and X-gal/IPTG, when applicable. Plates were incubated at 37°C overnight. Transformation of NEB 5-alpha chemically competent cells was the same as above with the following modifications. 25 μl of cells corresponded to half of the recommended cell volume per transformation. The cells were placed at 42°C for 30 seconds and were allowed to recover in 450 μl of SOC medium. Transformation of NEB 5-alpha electrocompetent cells was performed following the manufacturer’s protocol.
For p426-SNR52p-gRNA.CAN1.Y-SUP4t self-closure experiments 1 ng of the original gRNA plasmid was used as template DNA in a 50-μl PCR reaction. PrimeSTAR Max polymerase (2× Master Mix, Takara Bio) was used to generate both altered plasmids. PCR reactions were digested with DpnI for ~2 hours at 37°C and purified with Nucleospin Gel and PCR Clean-Up kit (Macherey-Nagel). 1 μl of the purified product (125–150 ng DNA) was combined with 25 μl NEB 5-alpha chemically competent cells and transformation was performed as above. For the P_TRC gRNA pUC alteration, PCR was carried out using 0.4 ng of the original gRNA plasmid and PrimeSTAR Max polymerase in a 20-μl reaction. PCR product was purified (without DpnI digest). When 1 μl (38 ng) of the purified product was combined with 10 μl NEB 5-alpha chemically competent cells, ~1,000 colonies formed on an ampicillin plate.
To verify correct assembly of the plasmids (not including those in plasmid-alteration experiments), colony PCR was performed with Quickload OneTaq polymerase (2× Master Mix, New England Biolabs) using primers outside of the insertion junctions (S2 Table). For Sanger sequencing, colonies were cultured in LB medium containing the appropriate antibiotics and the DNA was isolated using a miniprep kit (Qiagen, Valencia, CA).