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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 *did not work.. might be RNase contamination and will test that. need to figure out how to remove RNase contamination. *1. try combining samples and cleaning with guanidine HCl (should destroy RNases, then IVT) *2. sephadex columns and fractioning. fragmentation assay for activity. test with IVT *3. try epicentre IVT kit. has RNase inhibitor *4. research on how to remove RNases during isolation. do everything the same as july 2, 2014 isolation? HEPES as buffer, TX100 not changed. no changes in cleavage buffer. samples. washed with .5% TX100, 1 M NaCl. eluted .05% TX100, 0.8 M NaCl 1. f1, undil tsase, 6 ng DNA 2. f1, 2X dil. tsase, 6 ng DNA 3. f1, 4X dil. tsase, 6 ng DNA 4. f1, 8X dil. tsase, 6 ng DNA 5. f1, 16X dil. tsase, 6 ng DNA 6. f2, undil tsase, 6 ng DNA 7. f2, 2X dil. tsase, 6 ng DNA 8. f2, 4X dil. tsase, 6 ng DNA 9. f2, 8X dil. tsase, 6 ng DNA 10. f2, 16X dil. tsasa, 6 ng DNA 11. 6 ng pure DNA [[File:ZhangLab 2 2014-09-09 18hr 09min-labeled.jpg|600px]] *'''9/10/14 Testing transposase for RNase contamination''' *incubated 8/8/14 #16 IVT RNA with transposase. *looks like there is still RNase contamination. solution composition 5,6,7 uL 1X lysis buffer 1 or 2 uL RNA (8/13/14 #11 RNA, positive control nextera 6 ng DNA) ______________ 8 uL total *incubated for ~17 hours at 37C [[File:ZhangLab 2 2014-09-10 12hr 56min-labeled.jpg|600px]] *'''9/19/2014 update, transposase inactivation with amicon filters''' *In R1 isolation of transposase, nearly all transposase was inactivated by the amicon filters, thus leading to a repeat of the procedure. After incubation with transposome complexes genomic DNA bands were still present with no diltion, thus indicating there is no nuclease contamination. *only transposase used in reactions. not transpsosome complex. 1. This can show nuclease contamination was not present in R1. [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-6-19 R1 transposome complexes do not fragment genomic DNA, after inactivation with amicon filters] 2. Nuclease contamination is present above 10 kd in R4 f2, since transposase would be inactivated. *Spun in amicon filters 4X. increased speed since there was not much elution 1. 3k rcf, 10 mins 2. 3k rcf, 10 mins 3. 4k rcf, 10 mins 4. 4k rcf, 10 mins *Transposase only, reaction at 55C for 5 mins, followed by qiagen protease digestion, 50C for 10 mins, 70C for 20 mins. *reaction composition 1 uL 5X custom tagmentation buffer (buffer D6) 1 uL pure Jurkat DNA (6 ng) 1 uL amicon flowthrough or not. 1 uL 2X Lysis buffer 1 uL H2O __________ 5 uL total samples. all with 6 ng DNA Jurkat DNA. transposase only, no transposon 1. R4 f2, 2 uL undil. >30 kd, elution 2. R4 f2, 1 uL undil. >30 kd, elution 3. R4 f2, 2X dil. >30 kd, elution 4. R4 f2, 4X dil. >30 kd, elution 5. R4 f2, 8X dil. >30 kd, elution 6. R4 f2, 2 uL undil. <30 kd, elution 7. R4 f2, 1 uL undil. <30 kd, elution 8. R4 f2, 2X dil. <30 kd, elution 9. R4 f2, 4X dil. <30 kd, elution 10. R4 f2, 8X dil. <30 kd, elution 11. 6 ng pure DNA control *was approximately 2X concentrated in amicon filter. *since only nucleases would be contributing to DNA degradation, contamination is present. Thus there was almost no contamination in R1. Repeat procedure and do everything as exactly in R1 **binding to beads at RT **columns washing, bead washing at RT **hepes instead of tris **same buffer compositions. **Rnase zap treatment/autoclave all glassware **Order Molecular biology grade reagents (DNase, Rnase, Protease tested and free) [[File:ZhangLab 2 2014-09-19 19hr 09min-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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