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===IVT Protocol=== *If need to make more transposome, do first 2 steps. If not goto step 3. '''Generation of t7tspn-top2 transposome''' 1. annealing of ME sequence to T7 transposon sequence **a. Make 100 uM stock solution of t7tspn-top2(8/22) and T7tspn-NoP-bot(8/22). **b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 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 20 minutes at RT 0.5 uL of annealed transposon 0.5 uL of 100% sterile glycerol 1.0 uL of Ez-TN5 transposase *store at -20, is good for a year '''Generation of custom barcode (3' side of fragments)illuminia adaptor transposome''' 1. annealing of ME sequence to Nextera transposon sequence **a. Make 100 uM stock solution of (sc1-T7tspn-NoP-bot, "pMENTS") and (sc-ILA-adaptor). **b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 1 minutes. Oligo's now at 10 uM in 50 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 20 minutes at RT 1.25 uL of annealed Nextera transposon 1.25 uL of 100% sterile glycerol 2.50 uL of Ez-TN5 transposase *store at -20, is good for a year 3. Prepare samples, lyse cells with lysis buffer CELL WASHING - (wash cells with PBS) *1. count cells, spin down all cells at 250-500 g for 5 minutes *2. wash with 1X PBS, centrifuge 250-500 g for 5 minutes, resuspend to XXXX cells/uL *3. Aliquot cells to sample tubes CELL LYSIS *1. dilute LB to 2X, or as needed (from 10X stock) *2. refrigerate or warm LB or keep at RT *3. add lysis buffer to cell aliquots, mix briefly (mineral oil optional) *4. transposition ready LYSIS BUFFER NOTES *Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O. 4. transposition reaction *add the below into one tube and incubate at '''37C FOR 30 mins.''' 1.0 uL washed cells 1.0 uL 2X Lysis Buffer 1.0 uL 5X custom tagmentation buffer (buffer D6) 1.0 uL prepared transposomes 1.0 uL Nuclease free water ___________ 5.0 uL total solution 5. Protease digestion of transposase, protease inactivation To each tube, add: 1 uL Qiagen Protease, use 1 uL of 11.1 ug/uL (.5 AU/ml) protease (dilute 5.5 uL 20 ug/ul with 4.5 uL N-H2O) Incubate: 50C 10 minutes, 70C 20 minutes 6. Fill in reaction *Add 1 uL 5X taq polymerase, run at 72C for 3 minutes. (OR 6 uL taq2X) (same as nextera) 7. Maxiscript (Ambion) T7 Protocol, IVT *DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification. a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) on ice, but keep 10X transcription buffer at room temp b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!! bring to 20 uL with Nuclease free water X uL DNA template (list 1 ug) 2 uL 10X Transcription Buffer 1 uL 10 mM ATP 1 uL 10 mM CTP 1 uL 10 mM GTP 1 uL 10 mM UTP 2 uL T7 Enzyme Mix b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide) 8. Clean with Zymo clean and concentrator now *elute samples in 10 uL of N-H2O *quanitate with Qubit or on TBU gel. '''AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO''' '''Results''' *TBU after IVT *still is contamination. though worked like it did with R2. samples. 1. R6 f1, undil tsase, 6 ng DNA 2. R6 f1, 2X dil. tsase, 6 ng DNA 3. R6 f1, 4X dil. tsase, 6 ng DNA 4. R6 f1, 8X dil. tsase, 6 ng DNA 5. R6 f1, 16X dil. tsase, 6 ng DNA 6. R6 f2, undil tsase, 6 ng DNA 7. R6 f2, 2X dil. tsase, 6 ng DNA 8. R6 f2, 4X dil. tsase, 6 ng DNA 9. R6 f2, 8X dil. tsase, 6 ng DNA 10. R6 f2, 16X dil. tsasa, 6 ng DNA 11. nextera, 6 ng pure DNA [[File:ZhangLab 2 2014-10-15 15hr 16min-labeled.jpg|600px]] [[File:ZhangLab 2 2014-10-16 17hr 54min-labeled.jpg|600px]] *Example of Tn5 band from gosta [[File:ZhangLab 2 2014-06-11 Gosta SDS-PAGE gel image.png|600px]]
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