High throughput mini-BAC DNA isolation: Protocol for four 96well plates: Day1: 1. Add 500µl of LB to each well of a square well block using the multichannel stepper pipet. 2. Inoculate 1µl of the glycerolstock to each well and seal with a Breathseal Sheet 3. Shake at 37°C for 8h with 350rpm 4. Add 1000µl LB to a new 96 well block and inoculate 5µl of the starter culture and seal with a Breathseal Sheet 5. Shake at 37°C for 18h with 350rpm. Day2: 6. Centrifuge for 7min at 3200 rcf 7. Remove supernatant by inverting the square well blocks above a waste container and remove remaining liquid on a paper towel. 8. Check the presence and absence of bacterial pellets. Use a internal control for a contamination check. 9. Add 30µl GTE, and carefully resuspend the pellet by pipetting 20 times up and down with a multichannel pipette and transfer the liquid into a new non skirted 96 well Microplate. 10. Add 60µl freshly prepared 0,2M NaOH, 1%SDS to each well of plate one. 11. Immediately after finishing one plate, seal it with a Micro-Amp Clear Seal and invert it very, very smoothly for two times. 12. Start to countdown 4min30sec when having inverted the first plate. 13. After 3min and 15sec centrifuge plate1 and 2 in order to spin down the liquid in the centrifuge (2000rpm for one second) 14. After 4min 30 sec start to add 50µl 7,5M Ammoniumacetate to each well of plate one, seal the plate again with a new Micro-Amp Clear Seal and carefully (!) invert the plate immediately after pipetting and put it on ice. 15. Start with the other microplates and repeat from step 11. 16. After finishing the last plate, incubate for 10 minutes on ice. Invert the plates smoothly after 3 minutes of incubating. 17. After 10min centrifuge all plates at 3200 rcf for 30 minutes at room temperature in the Beckmann Allegra centrifuge. Use the stacking tools when placing the microplates in the centrifuge. 18. Place a new 96-wells PCR plate in a rack, mount it on top of the each centrifuged old ones (A1 on A12) and quickly invert it. 19. Centrifuge again for 30min 3200rcf at room temperature. 20. Invert a second time as described at step 19 on a new non skirted 96 well Microplate. 21. Add 70µl isopropanol to each well, seal it with a tape pad and invert it very smoothly for three times. 22. Centrifuge again for 30min at 3200rcf and invert in order to remove supernatant (now the DNA is in the pellet). 23. Add 50µl 70% ethanol and seal it with a Micro-Amp Clear Seal as above 24. Centrifuge at 6000 rcf for 10min at room temperature 25. Invert to remove supernatant, beat several times against a clean tissue in order to completely remove the residual ethanol. Leave the plates inverted on a fresh tissue 26. Add 25µl MilliQ-water to each well, seal with an Adhesive PCR Foil Seal and put them in the refrigerator overnight to solve the DNA. Day 3 Exonuclease Treatment: 27. Resuspend Exonuclease in 225µl solvent according to the manual of the Large-construct kit. 28. Add to each well 5µl of the following mix: 3µl NEB1, 0,6µl Exonuclease, 0,9µl ATP, 0,15µl RNase and 0,35µl H2O and mix carefully by smoothly pipetting five times up and down. 29. Incubate the plates at 37°C for 4-12h in a PCR machine. 30. Inactivate the exonuclease by heating up the reaction to 75°C for 10min. Random amplification of BAC-DNA (DOP-PCR) •Perform for each BAC/PAC-sample three Hot Start PCR reactions according this pipetting schedule: Mix A 1 reaction: 10x reactionbuffer 3.5 μl MgCl2 2.0 μl dNTP's (4x25mM) 0.32 μl DOP primer (100 pmol/l) 0.8 μl BAC/PAC-DNA 1 μl Milli Q water 27,4 μl ----------+ 35 μl Mix B 10x reactionbuffer 0.5 μl Taq-polymerase 0.5 μl Milli Q water 4 μl ----------+ 5 μl •Add Mix B to Mix A during the initial denaturation step of the PCR. The PCR is performed in a AB-thermal Cycler 9700 or in a 96wells MJ-Cycler using this program: 94°C 3 min. 94°C 1.5 min. 30°C 2.5 min. 10 cycles Ramp to 72°C at 0.1 °C per second 72°C 3 min. 94°C 1.0 min. 62°C 1.5 min. 30 cycles 72°C 2.0 min. 72°C 8.0 min. 12°C hold •Concentrate the PCR-products by precipitation: -Pool the DOP1, DOP2 and DOP3 products. -Add 1 μl of Glycogen and mix. -Add 1 μl of 3M NaAc and 240 l ethanol. -Spin 30 min. on 6000g in the Beckman Bench top Centrifuge. -Wash with 100 μl 70% ethanol -Spin for 10 min. on 6000g. -Dry the DNA-pellet by spinning the plates upside down on 600rpm for 30s. -Dissolve the DNA-pellet in 18 μl spotting buffer. Alternatively it is possible to concentrate the pooled DOP-PCR products by filtrating through a MilliPore Multiscreen PCR filter plate, according to the manual supplied with the kit. •The amplified BAC/PAC-DNA is now ready for arraying. Array printing 1. Dissolve oligonucleotide probe or PCR product in the appropriate spotting solution to obtain the recommended final probe concentration: DNA Probes Final Spotting Concentration Oligonucleotides 10 - 20 μM PCR products 0.1 – 1 mg/ml 2. Transfer an appropriate volume of probes to a microtiter plate. 3. Set up the arrayer according to the manufacturer’s recommendations. If you were previously using slides that were thicker than 1.0 mm, for optimal spotting you may need to re-calibrate the distance between the slide surface and the spotting pins. 4. Print the substrates at 40-50% relative humidity at 20 to 25°C. Dissolve oligonucleotide probe or PCR product in the appropriate spotting solution to obtain the recommended final probe concentration: DNA immobilization 1. Incubate printed microarray slides in humidity chamber at room temperature for 30 min (see appendix for details of how to prepare this chamber) to ensure quantitative immobilization. 2. Proceed to washing.
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Description
- Slide E from Scott-Nexterion (germany) - Epoxysilane coated - Printing of arrays using Biorobotics Microgrid II printer with 48 nanopins(www.matrixtechcorp.com) - The arrays consist of 48 subgrids. Each BAC-clone is printed 4 times. Replicate spots are in same/ different subgrids.
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