Functional Genomics Research Stream

Transcription

1 Functional Genomics Research Stream Research Meeting: February 14, 2012 Nucleic Acid Preparation & Gel Electrophoresis

2 If you like FRI... you should Like FRI

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4 College of Natural Sciences- Career Design Center. utexas-cns/student/ Upload resumes, find out about job opportunities.

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6 Laboratory Issues Bulb in spectrophotometer replaced YPD contaminations Check Results Central often for tips/ feedback from mentors and myself

7 Genomics Research Agenda IV Enzymatic Assays III Molecular Preparations III Aseptic Cell Culture II Safety and Basic Operations I

8 Sample Purification Separating What You Want... From What You Do Not Want Use Case: Cell Matter & Nucleic Acids Two Major Types: 1. Column Purification 2. Phase Separation by Centrifugation

9 Column Purification Common Technique in Many Kits

10 Phase Separation by Centrifugation Spinning Rotor Applies Centrifugal Force Sample is Separated or Pelleted Current Protocols Essential Laboratory Techniques, Wiley

11 Nucleic Acid Preps 1. Liquid Culture 2. Cell Spin Down, Re-suspension 3. Cell Lysis 4. Add Organic Solvent 5. Phase Separation 6. Retrieve Aqueous Phase 7. Precipitation of Nucleic Acid 8. Pellet Nucleic Acid, Re-suspend

12 Yeast Genomic DNA Extraction Resuspend yeast cells in lysis buffer containing Tris, SDS and EDTA Fragment cell wall/membrane by vortexing. Spin down debris, phenol/chloroform extract lysate - isolate genomic DNA enzyme-explorer/learning-center/lysing-enzymes.html

13 Yeast Genomic DNA Extraction Phenol / Phenol:Chloroform Method How does it work?

14 Yeast Genomic DNA Extraction Phenol / Phenol:Chloroform Method How does it look? Denatured proteins at interphase High salt and acidic conditions- DNA to organic layer Chloroform sharpens interphase band Aqueous phase Interphase Organic phase

15 Phase Separation: Caution proteins nucleic acids aqueous phase Do Not Get Greedy interphase organic phase

16 Phase Lock Tubes aqueous phase gel layer organic phase

17 DNA-red Proteinblue Yeast Nucleic Acid Extraction Phase lock tubes

18 1. Transfer cells to 1.5 ml eppendorf tube. This protocol assumes you are starting with, oh, let us say 20 ml worth of cell culture that has been spun down in the large PAI 2.14 centrifuge and then re-suspended in a small amount (~1 ml of YPD). This 1 ml is then transfered to a 1.5 ml eppendorf tube. 2. Spin down at 2000 rpm for 2 minutes. 3. Decant YPD from eppendorf tube. 4. Re-suspend cells in 1 ml sterile water. 5. Spin down at 2000 rpm for 2 minutes. 6. Decant water from eppendorf tube. 7. Re-suspend cells in 500 µl lysis buffer. 8. Vortex cells at full speed for two minutes. 9. Spin down at 2000 rpm for 2 minutes. 10. Transfer the liquid phase to a 1.5 ml eppendorf tube. 11. Add 1 ml (25:24:1) Phenol:Chloroform mixed with Isoamyl Alcohol. 12. Vortex for one minute. 13. Prepare a heavy (yellow) or light (green) phase lock tube by spinning it at 12,000 rpm for 2 minutes. 14. Transfer cell mixture to heavy (yellow) or light (green) phase lock tube. (do not vortex phase lock) 15. Spin heavy (yellow) or light (green) phase lock tube at 12,000 rpm for 5 minutes. 16. Decant supernatant into clean 1.5 ml eppendorf tube. 17. Add 250 µl 5M ammonium acetate. 18. Add 900 µl 95% (or 100%) ice cold ethanol. 19. Precipitate for 15 minutes at -20 C. 20. Spin DNA pellet down full speed for 5 minutes. 21. Wash pellet with 500 µl of 70% ethanol. 22. Spin DNA pellet down full speed for 5 minutes. 23. Allow to dry, re-suspend in ~50 µl of autoclaved water. 24. Measure concentration with NanoDrop. culture cell spin down resuspend cell lysis phase separation aqueous phase precipitation pellet

19 The Steps to Prep culture cell spin down resuspend cell lysis phase separation aqueous phase precipitation pellet

20 Yeast Nucleic Acid Extraction RNA precipitation How does it work? Positively charged salt ions bind RNA backbone Reducing ionic strength of solution (adding alcohol) precipitates bound RNA Na + Na + Na + Na + problem_sets/large_molecules/08t.html

21 pellet Total RNA, #44B TWHITE, 2/14/12

22 Nucleic Acid Preparation Evaluation Success Determined By: Quantity How much do we have? Quality How pure is it? How intact is it? How can we answer these questions?

23 NanoDrop ND-1000 Quantity & Quality Evaluation Possible

24 Open Questions Why do we wash precipitated nucleic acids with ~70% ethanol? What is the best blank solution to use on the nanodrop? What is meant by A260 or A280? What is the expected A260/A280 of DNA or RNA?

25 Gel Electrophoresis Nucleic acids are positively or negatively charged molecules? Charged molecules move toward or away from opposite charge?

26 Gels: Top View

27 Gels: Side View - +

28 Gels: Loading & Running

29 Gels: Result

30 University Leicester in Education

31 University Leicester in Education

32 Gels as Quality Evaluation Use a gel to tell us something diagnostically. Gels can also be used for sample preparation. Current Protocols Essential Laboratory Techniques, Wiley

33 Gels as Quality Evaluation

34 Lab Progress Continue Research Progress Report II & III Consider Timing, Constraints - Prepare Do Not Procrastinate Do Not Abuse Freedom & Responsibility Lab Open & Mentors Present 10am to 8pm (M, W, Th, F) No Tuesdays For Now... Research Resource Calendars...