Journal: Nature protocols
Article Title: Base-resolution stratification of cancer mutations using functional variomics
Figure Lengend Snippet: Confirmation of mutation entry clones by barcoded next-gen sequencing Products of the PCR mutagenesis reaction are cloned into the pDONR223 vector via Gateway BP reaction and transformed into competent DH5α bacterial cells. Transformants are spotted on LB + Spectinomycin plates to isolate single colonies. One plate is shown here as a representation. This scheme illustrates the steps involved in mutation sample preparation and next-generation sequencing confirmation. PCR amplification should be done for each mutant at the single colony level prior to sample preparation (illustrated by 96 well plates M1-M4). If a given gene has multiple mutations to clone ( e.g. , four as shown), these mutation PCR products should be separated into different pools (Steps 35-36). A unique barcode adaptor is added to each pool by ligation (Step 37). All pooled mutation PCR products are then sequence-verified by next-generation sequencing technologies (454-FLX, Illumina Solexa, etc) (Steps 38-41).
Article Snippet: Round-bottom 96-well plates (Corning, cat. no. 3788) 50 ml sterile reagent troughs (Corning, cat. no. 4871) Thin-walled 96-well PCR microtiter plates (Bio-Rad, cat. no. HSP-9601) 15 cm sterile petri dishes (Fisher Scientific, cat. no. 08-757-14) Aluminum foil microplate seal (Bio-Rad, cat. no. MSF-1001) 50 ml polypropylene tubes (BD Falcon, cat. no. 352070) Tissue culture 96 wells flat bottom plate (BD Falcon, cat. no. 353075) PIPETMAN P10, 1 to 10 µl (Gibson, cat. no. F144802) PIPETMAN P200, 50 to 200 µl (Gibson, cat. no. F123601) Corning™ 4084 P10 multichannel pipettes 1 - 10 µl (Thermofisher scientific, cat. no. 07-764-710) Corning™ 4085 P50 multichannel pipettes 5 - 50 µl (Thermofisher scientific, cat. no. 07-764-711) Corning™ 4085 P200 multichannel pipettes 20 - 200 µl (Thermofisher scientific, cat. no. 07-764-712) Corning® Stripettor™ Ultra Pipet Controller (Thermofisher scientific, cat. no. 4099) Thermocycler capable of handling 96-well plates (Bio-Rad, cat. no. T100™) Temperature controlled incubator (30°C) (Thermofisher scientific, cat. no. 15-103-0515) Temperature controlled water bath (42°C) (Thermofisher scientific, cat. no. FSGPD10) 1% E-Gel® 96 Agarose Gels (Thermofisher scientific, cat. no. G700801) E-gel Mother E-Base capable of running 96 samples (Thermofisher scientific, cat. no. EBM03) Temperature controlled shaker for 250 ml flasks and 50 ml tubes (MaxQ™ 6000 Shaker, Thermofisher scientific, cat. no. SHKE6000) Benchtop Centrifuge suitable for microplates and 50 ml tubes (Sorvall™ Legend™ XT/XF centrifuge, Thermofisher scientific, cat. no. 75-004-505) Plate shaker (Mixmate, Eppendorf, cat. no. 022674200) GENESYS™ 10S UV-Vis Spectrophotometer (Thermofisher scientific, cat. no. 840-208100) Vortexer (Thermofisher scientific, cat. no. 88880017TS) Centro XS3 Luminescence Microplate Reader (Berthold Technologies, cat. no. 46970) 1300 Series A2 Class II, Type A2 Bio Safety Cabinets (Thermofisher scientific, cat. no. 13-261-221) Optional: liquid handling robot with 96-well multichannel head and individual well cherry-picking capability (Tecan, cat. no. Fluent 780) Optional: BioRobot Universal System Robot with 96-well DNA miniprep capability (Qiagen, cat. no. 9001094)
Techniques: Mutagenesis, Clone Assay, Sequencing, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay, Sample Prep, Next-Generation Sequencing, Amplification, Ligation