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===Results=== *TBU samples after IVT, and before purification. cell samples have a slightly less smear then usual. Pure DNA samples look normal {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''intensity''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||5||2077.610062|| |- | ||2.5||1089.340032||in 20 uL |- | 1||65.11745272||25842.52077||1302.349054 |- | 2||51.66176753||20523.38061||1033.235351 |- | 3||165.8200441||65651.06196||3316.400882 |- | 4||196.650637||77838.64232||3933.012739 |- | |} *[[File:ZhangLab 2 2013-06-11 14hr 00min-labeled.jpg|600px]] *For first set of samples, ran AMPURE RNA beads as normal. with 20 uL of IVT reaction, added 20 uL of beads and ran through the procedure. *can see that most of the sample is still in the supernatent (flow though), during first step of beads purification. For the pure DNA sample some was eluted in the elution, however most of it is in the flow though. *Using first set of trial samples Samples: 2. 1K cells cells beads T1 4. 6 ng Pure DNA beads T1 6. NTC (no tspn) beads T1 *TBU on beads washes, elutions etc. **4/40 uL supernatent used **5 uL resuspended beads **1/10 uL elution used [[File:ZhangLab 2 2013-06-11 16hr 12min-labeled.jpg|600px]] *For second set of trial samples, first 30 uL of water was added to samples, and then 100 uL of beads for a total of 150 ul in solution. Rest of the beads purification protocol was followed as normal, with ~3 minutes used for drying the beads. *here most of the sample is in the elution for both pure DNA and where 1K cells were used. DNA band at ~80 bp is lost, which is what is wanted. The beads still retain some pure DNA sample (lane 5) probably because there is alot of sample. can even try a higher beads ratio. *second set of trial samples Samples: 1. 1K cells beads T2 3. 6 ng Pure DNA beads T2 5. NTC (no tspn) beads T2 *TBU on beads washes, elutions etc. **4/150 uL supernatent used **5 uL resuspended beads **1/10 uL elution used [[File:ZhangLab 2 2013-06-11 19hr 08min-labeled.jpg|600px]] *quantification of results. can see with T1 samples, especially pure DNA sample, most of the sample is in the supernatant. When the altered protocol was used with more beads, most of the sample was in the elution, with ~40-50% sample loss. {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''intsnity''' | align="center" style="background:#f0f0f0;"|'''total volume''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||5||2772.840083|||||||| |- | ||2.5||1662.26005||||total ng||originally in sample||loss |- | 1. beads flow through 2. 1K cells T1||9.771560806||4892.520146||38||371.3193106||1302.349054||71.49% |- | 2. beads flow through 4. 6 ng pure DNA T1||105.9414225||47614.25142||38||4025.774055||3316.400882||-21.39% |- | 3. beads flow through 6. NTC (no tspn) T1||-1.067640332||77.40000231||38||0||N/A|| |- | 4. beads after elution 2. 1K cells T1||2.957868861||1865.660056||5||14.78934431||1302.349054||98.86% |- | 5. beads after elution 4. 6 ng pure DNA T1||9.601221884||4816.850144||5||48.00610942||3316.400882||98.55% |- | 6. beads after elution 6. NTC (no tspn) T1||-1.40309568||-71.62000213||5||0||N/A|| |- | 7. 1 uL elute 2. 1K cells T1||3.638751823||2168.130065||10||36.38751823||1302.349054||97.21% |- | 8. 1 uL elute 4. 6 ng pure DNA T1||27.48280178||12760.42038||10||274.8280178||3316.400882||91.71% |- | 9. 1 uL elute 6. NTC (no tspn) T1||0.149899152||618.2700184||10||1.498991518||N/A|| |- | |||||||||||| |- | |||||||||||| |- | |||||||||||| |- | |||||||||||| |- | ||ng/ul||intensity||total volume (ul)|||||| |- | ||5||3113.460093|||||||| |- | ||2.5||2288.280068||||total ng||originally||loss |- | 1. beads flow through 1. 1K cells T2||-3.608182457||272.1400081||150||0||1033.235351||100.00% |- | 2. beads flow through 3. 6 ng pure DNA T2||9.117192613||4472.430133||150||1367.578892||3933.012739||65.23% |- | 3. beads flow through 5. NTC (no tspn) T2||-3.266135873||385.0400115||150||0||N/A|| |- | 4. beads after elution 1. 1K cells T2||8.192848833||4167.330124||5||40.96424416||1033.235351||96.04% |- | 5. beads after elution 3. 6 ng pure DNA T2||45.27912092||16408.47049||5||226.3956046||3933.012739||94.24% |- | 6. beads after elution 5. NTC (no tspn) T2||-4.005307933||141.0600042||5||0||N/A|| |- | 7. 1 uL elute 1. 1K cells T2||52.85895199||18910.36056||10||528.5895199||1033.235351||48.84% |- | 8. 1 uL elute 3. 6 ng pure DNA T2||240.3917933||80809.70241||10||2403.917933||3933.012739||38.88% |- | 9. 1 uL elute 5. NTC (no tspn) T2||33.8997552||12652.46038||10||338.997552||N/A|| |} *'''UPDATE 6/17/2013 RNA CONCENTRATION TESTING''' *used samples after beads purification. Pure DNA sample #8 was used for different RNA amounts. 1K cells was used as one sample. *TBE after barcode addition [[File:ZhangLab 2 2013-06-13 19hr 12min-labeled.jpg|600px]] *quantification for gel size selection {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''intensity''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||||5||3323.790099|||| |- | ||||2.5||1888.290056||for 55 ng sample|| |- | 49||1-7. 1X RnIII cells sample (528 ng RNA)||13.96048415||8468.900252||3.939691446|| |- | 50||2-8. No RnIII (200 ng RNA)||12.69292929||7741.070231||4.33312112|| |- | 51||3-8. 1XRnase III (0 ng RNA)||4.94235458||3290.690098||11.12829909|| |- | 52||4-8. 1X RnIII (100 ng RNA)||5.597439916||3666.840109||9.825920568|| |- | 53||5-8. 1X RnIII (200 ng RNA)||7.248066876||4614.630138||7.588230206|| |- | 54||6-8. 1X RnIII (200 ng RNA) NO BP||7.927220481||5004.600149||6.938119122|| |- | 55||7-8. 1X RnIII (400 ng RNA)||14.98146987||9055.15027||3.671201856|| |- | 56||8-8. 1X RnIII (800 ng RNA)||34.5530303||20293.1406||1.591756194|| |- | 57||9-8. 1X RnIII NTC sample||3.968443051||2731.470081||---|| |- | |||||||||| |- | ||||9||||49.01633961||total uL |- | ||||55||||9.169389934||dye to add |- | ||||495||||58.18572954||sum |- | |} *after gel size selection [[File:ZhangLab 2 2013-06-17 10hr 00min.jpg|600px]] *validation of gel size selection *selected for ~220-800 bp, decent smear. [[File:ZhangLab 2 2013-06-17 14hr 19min-labeled.jpg|600px]]
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