Extracting DNA Using Phenol-Chloroform. Reagents Needed. 1. phenol/chloroform/isoamyl alcohol (PCI) solution (25:24:1) DNase (RNase- and Protease-Free -.
Hawinkel, Werner Van Leekwijck. Bell Labs. Alcatel-Lucent. Antwerp, Belgium [email protected]. Jeroen Famaey, Wim Van de Meerssche,.
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One of the most important, but frequently overlooked, cell culture procedures is ... Photomicrographs (1000x) of VERO cells stained with Hoechst 33258 dye.
Introduction. One of the most important, but frequently overlooked, cell culture procedures is testing cultures for microbial contamination, especially mycoplasma.
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such as video/audio conferencing, replicated database, information distribution, resource discovery, and replicated web publishing, etc. However, implementing ...
Introduction. Plant materials are among the most difficult for high quality DNA extractions. The key is to properly prepare the tissues for extraction. In most cases ...Missing:
2% (w/v) hexadecyltrimethylammonium bromide (CTAB). 2.0 g. Extraction Buffer A (EBA). Per 100 mL. 100 mM Tris (pH 8.0) (Use 1 M stock). 10 mL.
Introduction. Plant materials are among the most difficult for high quality DNA extractions. The key is to properly prepare the tissues for extraction. In most cases ...
Thomas Building, DE-780. 206.667.4289 ..... evaluated and the procedure is adjusted to make sure the best results will be obtained. ..... For more information on the topics listed below, please check the websites or publications listed below.
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Cite this article as: Healey, A., Furtado, A., Cooper, T. et al. Plant Methods ..... Kasem S, Rice N, Henry RJ: DNA Extraction from Plant Tissue. Plant Genotyping II: ...
Remove ~180 µL of the top aqueous solution and place into a new tube, TUBE 2. ... TUBE 2: Chloroform Back Extraction (the following steps are to be performed in ... applicable license terms at http://www.pacificbiosciences.com/licenses.html.
PacBio SampleNet – Shared Protocol Please note: the shared protocols described herein may not have been validated by Pacific Biosciences and are provided as-is and without any warranty. Use of these protocols is offered to those customers who understand and accept the associated terms and ® conditions and wish to take advantage of their potential to help prepare samples for analysis using the PacBio RS system. If any of these protocols are to be used in a production environment, it is the responsibility of the end user to perform the required validation.
Extracting DNA Using Phenol-Chloroform Reagents Needed 1. phenol/chloroform/isoamyl alcohol (PCI) solution (25:24:1) DNase (RNase- and Protease-Free Molecular Biology grade), pH 7.8-8.2 Important: acidic pH makes the DNA go into the phenolic layer while RNA goes into the aqueous layer Suppliers http://www.sigmaaldrich.com/catalog/product/sigma/77617?lang=en®ion=US http://products.invitrogen.com/ivgn/product/15593031 http://www.fishersci.com/ecomm/servlet/fsproductdetail_10652_657663_29104_-1_0
Procedure TUBE 1: Phenol/Chloroform/Isoamyl Alcohol Extraction 1. Start with 200 µL of material and a tube (label as TUBE 1). If necessary, bring the volume up to 200 µL using the Elution Buffer (“EB”) above. 2. Add an equal volume of the phenol/chloroform/isoamyl alcohol solution to TUBE 1. 3. Vortex TUBE 1 vigorously for 1 minute. 4. Spin TUBE 1 solution at high speed for 5 minutes. 5. Remove ~180 µL of the top aqueous solution and place into a new tube, TUBE 2. Avoid picking up any of the phenol/chloroform/isoamyl alcohol phase. 6. Add 200 µL of EB to TUBE 1. 7. Vortex TUBE 1 vigorously for 1 minute. 8. Spin TUBE 1 solution at high speed for 5 minutes. 9. Remove as much of the top aqueous solution as possible from TUBE 1 without picking up any of the phenol/chloroform/isoamyl alcohol phase. Add the solution to TUBE 2.
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PacBio SampleNet – Shared Protocol TUBE 2: Chloroform Back Extraction (the following steps are to be performed in TUBE 2) 10. Add equal volumes of the chloroform/isoamyl alcohol solution to TUBE 2. 11. Vortex TUBE 2 vigorously for 1 minute. 12. Spin TUBE 2 solution at high speed for 5 minutes. 13. Remove as much of the top aqueous solution as possible and place into a new tube, TUBE 3. Avoid picking up any of the chloroform/isoamyl alcohol phase. TUBE 3: Ethanol Precipitation (the following steps are to be performed in TUBE 3) 14. Add NH4OAc to a final concentration of 0.75 M. 15. Add 1 µL of glycogen (20 µg). 16. Mix solution well. 17. Add 2.5X volume of 100% ethanol and mix well. 18. Incubate at 20 °C (optional). 19. Spin for 20 minutes in a 4 °C centrifuge at top speed. 20. Decant supernatant carefully without disturbing the pellet. 21. Wash by adding 300 µL of 80% EtOH and vortex 3 times. 22. Spin for 15 minutes in a 4 °C centrifuge at top speed. 23. Decant supernatant carefully without disturbing the pellet. 24. Repeat steps 21 – 23 for a second 80% EtOH wash. 25. Quick spin on table top centrifuge to draw residual EtOH to the bottom. 26. Remove residual EtOH with a P20 pipette. Be careful not to disturb the pellet. 27. Air dry for 1-2 minutes. 28. Re-suspend in appropriate volume EB (based on input amount).
