CTR:Notebook/PIRE/2014/07/09

PIRE RAD Library Preps

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RAD Library Prep - Olive Sunbirds Plate 2, Little Greenbuls Plates 1 & 2 96 well plate of 50ng of DNA 10uL For Digestion & Ligation steps: Sunbirds Plate 2 - 2014-07-09, Sork thermocycler Greenbuls Plate 1 - 2014-07-11, Tony thermocycler Greenbuls Plate 2 - 2014-07-14, Sork thermocycler Digestion Mix and add to each well: 0.68 uL water 1.20 uL NEBuffer 4 0.12 uL SbfI-HF Incubation: 37 degrees for 60 minutes 65 degrees for 20 minutes P1 adapter ligation Add 2 uL indexed P1 adapter (10nM) to each well Mix and add to each well: 1.28 uL water 0.40 uL NEBuffer 4 0.16 uL ATP 0.16 uL T4 Ligase Incubation: 20 degrees for 60 minutes 65 degrees for 20 minutes The rest of the steps were done with all 3 libraries: Clean up - 2014-07-15 Take 5 uL from each well and combine to 1.7 mL tube AMPure bead clean up: Use ~460 uL beads to DNA (1:1) 2 washes of 800 uL 80% EtOH Elute in 100 uL LowTE Sonication - 2014-07-15 BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power 2μL of sheared DNA in each well 2014-07-16: Blunt End Repair Mix: 50.0 uL fragmented DNA 5.5 uL LowTE 3.0 uL End Prep Enzyme Mix 6.5 uL End Repair Reaction Buffer Incubation: on John Block B 20 degrees for 30 minutes 65 degrees for 30 minutes P2 adapter ligation Add to mix: 15.0 uL Blunt/TA Ligase Master Mix 2.5 uL P2 RAD adapter (5 uM) 1.0 uL Ligation Enhancer Incubation: on John Block B 20 degrees for 15 minutes Size selection Add 16.5 uL water for a total of 100 uL AMPure bead size selection: 45 uL beads to remove large fragments 25 uL beads to remove small fragments 3 washes of 200 uL 80% EtOH Elute in 20 uL LowTE PCR amplification Mix: 5 uL DNA 25 uL NEBNext High Fidelity 2x PCR Master Mix 18 uL water 1 uL P1 adapter primer (25 uM) 1 uL P2 adapter primer (25 uM) PCR cycle: on John Block B 98 degrees for 30 seconds 15 cycles of: 98 degrees for 10 seconds 65 degrees for 30 seconds 72 degrees for 30 seconds 72 degrees for 5 minutes 1) 1 μL Sunbird Plate 2 template, 2) 5 μL Sunbird Plate 2 PCR product 3) 1 μL Greenbul Plate 1 template, 4) 5 μL Greenbul Plate 1 PCR product 5) 1 μL Greenbul Plate 2 template, 6) 5 μL Greenbul Plate 2 PCR product → PCR products do not look 2x as bright as the template. No significant amplication. Troubleshooting Tried re-prep of leftover sheared DNA (Blunt End Repair → PCR), but no difference Used old P1 primer in amplification → Works. Could mean that P1 adapter ligation failed and only fragments with P2 adapters on both ends are amplifying. Start with leftover DNA from Greenbuls Plate 2 and re-shear to see if overshearing caused lack of P1 adapter to most fragments. 1) Pre-cleaned DNA (post-P1-ligation), 2) Cleaned DNA, 3) Sheared DNA 1st shearing step looks good - proceed to Blunt End Repair → PCR 1) 1μL template, 2) 5μL PCR product → Still no amplification. Sonication is not the issue. Test Sunbirds Plate 1 template and replicate PCR, and check if failing templates will amplify with only P2 Primer 1&2) Sunbirds Plate 1, 3&4) Greenbuls Plate 1 (from re-sheared template) → PCR with good template still works. P2 primer only does not work at all Re-prep Greenbuls Plate 1 using leftover DNA from plate Used new SbfI-HF tube and new T4 ligase. 2014-07-31: Digestion: RADIGEST on Tony Ligation: RADLIG on Tony Used adapters from original Berkeley plate (undiluted) Clean up Sonication: 8 cycles of 15 seconds on, 90 seconds off, High Power Do not have have gel image for 2nd shearing attempt (4 additional cycles), but looked better than first. Because final attempt with additional 2 cycles did not look right (more large fragments), decided to continue with subsequent steps to avoid further sonication problems. End Repair: NEBENDRP on John Block B P2 Ligation: NEBLIGAT on John Block B 2014-08-01: Size Selection: 40μL beads to remove large fragments (decreased amount to reduce loss of DNA), 25μL to remove small PCR: NEBPCR on John Block B 1μL template, 2μL 100bp ladder, 5μL PCR product →Successful amplification! Clean up: 45 μL beads eluted in 30 μL LowTE PicoGreen: 8.44 ng/μL Re-prep Greenbuls Plate 2 Used same SbfI-HF and ligase from previous library. New DNA plate prepped 2014-08-05 Digestion: RADIGEST on Tony 2014-08-06 Ligation: RADLIG on Tony 2014-08-06 Adapters from original Berkeley plate Clean up 2014-08-06 2014-08-07: Sonication 8 cycles of 15 seconds on, 90 seconds off, high power. 2μL of ladder and sheared DNA. Blunt End Repair: NEBENDRP on John Block B P2 Adapter Ligation: NEBLIGAT on John Block B Size selection: 40μL to remove large fragments, 25μL to remove small fragments PCR: NEBPCR on John Block B 1) 1μL template. 2) 2μL ladder. 3) 5μL PCR product →No amplification Re-prep Sunbirds Plate 2 and Greenbuls Plate 2 Re-prep from existing plates. Received new SbfI-HF from NEB. Digestion 2014-08-13 Sunbirds: RADIGEST on Sork Greenbuls: RADIGEST on Tony Ligation 2014-08-13 Sunbirds: Used Berkeley adapter plate and old T4 ligase. RADLIG on Sork Greenbuls: Used mostly Miller adapter plate and new T4 ligase. RADLIG on Tony Bead Cleanup 2014-08-13 Sonication 2014-08-14 8 cycles followed by 4 + 2 + 2 additional cycles (16 total) Blunt End Repair 2014-08-14: NEBENDRP on John Block B P2 Adapter Ligation 2014-08-14: NEBLIGAT on John Block B. Used new tube of P2. Size Selection 2014-08-15: 40 μL + 25 μL beads PCR 2014-08-15: NEBPCR on John Block B 1) 1 μL Sunbird plate 2 template; 2) 5 μL Sunbird plate 2 PCR product; 3) 2 μL 100bp ladder; 4) 1 μL Greenbul plate 2 template; 5) 5 μL Greenbul plate 2 PCR product →Using new SbfI-HF seems to work. New enzyme and ligase is ideal! Clean up 2014-08-19: 45μL beads eluted in 30μL LowTE PicoGreen Sunbirds Plate 2: 4.74 ng/μL Greenbuls Plate 2: 7.79 ng/μL BioAnalyzer results from all 3 libraries 1) Sunbirds Plate 2; 2) Greenbuls Plate 1; 3) Greenbuls Plate 2