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===Protocol=== *If need to make more transposome, do first 2 steps. If not goto step 3. '''Generation of sc1-T7iBR-IdxXX transposomes''' 1. annealing of ME sequence to T7 transposon sequence **a. Make 100 uM stock solution of sc1-T7iBR-IdxXX and sc1-T7tspn-NoP-bot. **b. Incubate 2.5 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 1 minutes. Oligo's now at 10 uM in 25 uL. **c. cool to RT at 0.1 C/s 2. transposome complex generation, run controls!!! *'''add the below components into one tube and incubate for 30 minutes at 37C (DID THIS TIME)''' 1. Add 0.5 uL of 100% sterile glycerol to tube 2. Add 0.5 uL of annealed transposon to the 100% sterile glycerol and mix well 4. Add 1.0 uL of '''DILUTED''' Ez-TN5 transposase to well. *store at -20, is good for a year 3. Prepare samples, lyse cells with lysis buffer 4. transposition reaction Add all components and incubate at 55C for 6 minutes 1.0 uL 5X Custom Tagmentation buffer 2.0 uL lysed cells/pure genomic DNA 1.0 uL (or .6 uL undiluted) prepared T7 transposomes (MAKE SURE TO ADD LAST) (5X diluted) 1.0 uL Nuclease free water ___________ 5.0 uL total solution 5. Protease digestion of proteins/transposase To each tube, add: 1 uL Proteinase K Incubate: 50C 10 minutes, '''Did not do 70C inactivation of protease'''
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