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===Validation of results=== *Successfully completely fragmented 6 ng of DNA. *R6 f1 0.91 average abs; 10.52 uM, 3.2 mL total, final of 1.1 mL, 45% concentration from 20% glycerol to 50%, '''15.25 uM''' *R6 f2 0.83 average abs; 9.59 uM, 3.2 mL total, final of 1.2 mL, 42% concentration from 20% glycerol to 50%, '''13.58 uM''' *calculation for uM. 100 uM/8.65 = X uM/abs '''UPDATE 10/23/2014''' *bradford assay with standard curve. About the same as calculation with nanodrop when accounting for concentrating after doing dialysis into storage buffer, so values are accurate. {| {{table}} | align="center" style="background:#f0f0f0;"|R6 f1 | align="center" style="background:#f0f0f0;"|0.77365 | align="center" style="background:#f0f0f0;"|absorbance |- | R6 f2||0.80245||absorbance |- | |||| |- | R6 f1||0.743407877||ug/ul |- | R6 f2||0.791471963||ug/ul |- | |||| |- | '''R6 f1'''||'''14.02656372'''||'''uM''' |- | '''R6 f2'''||'''14.93343326'''||'''uM''' |- | |||| |- | ||1.42||mg/ml |- | ||||ug/ul |- | ||||g/l |- | |||| |- | |} [[File:2014-10-23 R6 tn5 bradford standard curve.png|600px]] '''10/14/2014''' *'''incubation with transposase only to test nuclease contamination''' *looks like there is still nuclease contamination *with 6 ng DNA *incubated reaction at 55C for 5 mins, followed by qiagen protease digestion, 50C for 10 mins, 70C for 20 mins. *reaction composition 1 uL pure Jurkat DNA (6 ng) 1 uL 2X Lysis buffer 1 uL 5X custom tagmentation buffer (buffer D6) 1 uL H2O 1 uL transposase __________ 5 uL total 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 f2, undil tsase, 6 ng DNA 6. R6 f2, 2X dil. tsase, 6 ng DNA 7. R6 f2, 4X dil. tsase, 6 ng DNA 8. R6 f2, 8X dil. tsase, 6 ng DNA 9. 6 ng pure DNA [[File:ZhangLab 2 2014-10-14 14hr 11min-labeled.jpg|600px]] [[File:ZhangLab 2 2014-10-14 14hr 13min-invert-labeled.jpg|600px]] *'''10/1/14 Testing transposase for RNase contamination''' *incubated 9/12/14 #11 IVT RNA with transposase. *looks like there is no RNase contamination solution composition 5,6,7 uL 1X lysis buffer 1 or 2 uL RNA (9/12/14 #11 RNA, positive control nextera 6 ng DNA) ______________ 8 uL total *incubated for ~17 hours at 37C '''11/13/14 UPDATE, TESTING TSOME CMPLEX FRAGMENTATION''' '''tsome complex testing R6f1''' with 6 ng DNA 1. dilute transposase to respective dilutions, 1X, 2X, 4X, 8X, 16X, 25X, 32X 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 (10 uM) 0.5 uL of 100% sterile glycerol 1.0 uL of Ez-TN5 transposase 3. make following reaction *reaction composition 1 uL pure Jurkat DNA (6 ng) 1 uL 2X Lysis buffer 1 uL 5X custom tagmentation buffer (buffer D6) 1 uL H2O 1 uL transposase __________ 5 uL total incubated reaction at 55C for 5 mins, followed by qiagen protease digestion, 50C for 10 mins, 70C for 20 mins. 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 f1, 25X dil. tsase, 6 ng DNA 7. R6 f1, 32X dil. tsase, 6 ng DNA 8. 6 ng pure DNA alone 4. run on TBE gel *lane 4 8X dilution probably wasn't pipetted correctly as 16X dilution causes more fragmentation. was pipetted by the undergrad. *also gel was left overnight at 4C in TBE in plastic bag since we ran out of sybr gold [[File:ZhangLab 2 2014-11-14 10hr 55min-labeled.jpg|600px]] [[File:ZhangLab 2 2014-11-14 10hr 56min-invert-labeled.jpg|600px]] '''10/2/2014 IVT testing of transposase'''
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