0805041236_dna.extraction by chathura

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									Genomic DNA Extraction and Biology Review Quantification

Objectives The objective of genomic potato DNA extraction using CTAB protocol is to obtain DNA that will be used for PCR amplification and microsatellite analyses.

Principles
DNA Extraction via CTAB Method

The CTAB method of DNA extraction is the best method of DNA extraction for obtaining good quality total DNA from many plant species. CTAB is a cationic detergent, which solubilizes membranes and forms a complex with DNA. After cell disruption and incubation with hot CTAB isolation buffer, proteins are extracted by chloroform/ isoamyl alcohol. The CTAB-DNA complex is precipitated with isopropanol. The DNA pellet resulting after centrifugation is washed, dried and redissolved. RNase treatment and ammonium acetate precipitation removes RNA and some polysaccharides, respectively.
DNA Quantification via Agarose Gel Electrophoresis

Electrophoresis through agarose (or polyacrylamide) gels is the standard method used to separate, identify and purify DNA fragments. The location of DNA within the gel can be determined directly by staining with low concentrations of the fluorescent dye ethidium bromide and visualizing with ultraviolet light.

Summer Genomics Workshop Protocols

Tuesday, June 22nd
Presentation on PCR (Amy) Presentation on Restriction Digest (Jack, Anthony) Finish solutions and buffers Prepare mortars and pestles for tissue grinding Agarose gel preparation for DNA quantification

Mortar and Pestle Preparation
Equipment and Reagents Mortars Spatulae Procedure 1. 2. 3. Wrap Spatulae in Foil, Autoclave and Freeze Retrieve Mortar and Pestle from Gel Doc room Place in -80oC freezer in Hibay Pestles Aluminum foil

DNA Quantification (Agarose Gel Preparation)
Equipment and Reagents 1x TAE Buffer SeaKem agarose Small weigh boat Gel comb Gel power supply Ethidium bromide Small plastic graduated cylinder Agarose only beaker Gel plate Gel Box

Gel aid
Procedure 1. 2. 3. 4. 5. 6. 7. 8.

Plastic wrap

Measure 150 mL into graduated cylinder Pour into glass beaker Weigh 1.5 g Agarose and add to beaker Microwave Beaker for 1 minute Let cool Set up gel plate in Gel aid Set up Comb Add 1.5 L EtBr **EtBr is a mutagen so be extremely careful and change gloves after addition to gel 9. Pour Agarose/ TAE Mixture into plate 10. Let solidify 11. Wrap in saran wrap and store overnight at 4oC

Summer Genomics Workshop Protocols

Principles
DNA Extraction via CTAB Method

The CTAB method of DNA extraction is the best method of DNA extraction for obtaining good quality total DNA from many plant species. CTAB is a cationic detergent, which solubilizes membranes and forms a complex with DNA. After cell disruption and incubation with hot CTAB isolation buffer, proteins are extracted by chloroform/ isoamyl alcohol. The CTAB-DNA complex is precipitated with isopropanol. The DNA pellet resulting after centrifugation is washed, dried and

Summer Genomics Workshop Protocols

Wednesday, June 23rd
Greenhouse orientation by Julie Mathern Tissue Collection for SSR project Lab meeting Tissue Grinding for SSR project

Tissue Collection
Equipment and Reagents Styrofoam Containers Dry Ice Liquid Nitrogen Procedure 1. 2. 3. 4. 5. Obtain dry ice from basement Go to greenhouse and find Julie or David Follow correct greenhouse suiting protocol Label plastic bags Collect 2-3 g of each type of tissue a. Collect Young and Healthy Leaves b. Follow Proper Collection Techniques (either pinching or razor) c. Avoid collecting stems, try to get only leaves 6. Back in the lab: weigh leaves a. Use each piece of foil to tare 7. Label each piece of foil with weight 8. Place foil wrapped leaves in liquid nitrogen 9. Write weights in lab notebook 10. Place foil in –80oC freezer

Summer Genomics Workshop Protocols

Tissue Grinding
Equipment and Reagents Label 50 mL Orange Capped Tubes Tube Rack Syrofoam Buckets Dewey Container for Liquid Nitrogen Procedure 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Clean bench area Retrieve dry ice and liquid nitrogen from basement Place dry ice in styrofoam container Label tubes Retrieve mortar, pestle and spatulae from freezer Retrieve leaves from freezer Place mortar and pestle in dry ice container Pour small amount of liquid nitrogen in mortar Put leaves into mortar Pour liquid nitrogen into mortar (about 1/2 full) Place orange capped tubes into liquid nitrogen container #2 Grind leaf tissue into fine powder Add liquid nitrogen as needed Place tube rack with orange capped tubes in liquid nitrogen in styrofoam container Dip spatula into liquid nitrogen Transfer leaf tissue into labeled tubes Place labeled tubes in –80oC freezer overnight

Step 8

Step 12

Step 16

Summer Genomics Workshop Protocols

Friday, June 25th
Presentation SSRs (Hanhui/ Wai) (Conference Room 5) Review primers for SSR project, complete worksheet DNA extraction for SSR project DNA quantification for SSR project

CTAB Extraction Buffer Preparation
Equipment and Reagents 50 mL Orange Capped Tubes (Sterile) 2X CTAB Buffer Procedure 1. 2. 3. 4. Clean bench area Label tubes by tissue name Pour 15 mL 2x CTAB into tubes Incubate in 65oC water bath (Room 2303) Permanent Marker Tube Racks

DNA Isolation
Equipment and Reagents Chloroform RnaseA Chloroform/ Isoamyl Solution Isopropanol 70% EtOH Sterile Black Topped Bottle Spatulae Bag of 50 mL Orange Capped Tubes Procedure 1. 2. 3. 4. 5. 6. 7. Retrieve Leaf Tissue from Freezer Transfer Leaf Tissue Into Extraction Buffer Tube Cap and Gently Invert to Mix Incubate in 65oC Water Bath a. Mix by Inversion every 20 minutes for 1 hour Clean-Up Materials a. Place all Disposable Items in Garbage b. Place all Reuseable Items at Emilio’s Bench Cool Tubes at Bench for 10 minutes Add 15 mL Chloroform (in hood) Permanent Marker 5 mL Glass Pipets Pipet Aid 5 mL Plastic Pipets Pipetman Pipet Tips

Eppendorf Tubes

Step 4

Step 7

Summer Genomics Workshop Protocols

Procedure con’t. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. Shake Tubes for 1 minute Centrifuge Tubes at 3,000 rpm for 15 minutes Step 8 Label 50 mL Orange Tubes Transfer Supernatant to New Tubes Add 30 µL RNaseA Incubate at 37oC for 1 hour Add 15mL Chloroform/ Isoamyl Solution (in hood) Shake Tubes for 1 minute Centrifuge Tubes at 3,000 rpm for 15 minutes Label 50 mL Orange Tubes Step 20 Transfer Propanol to Sterile Bottle Transfer Supernatant to New Tubes Add 18 mL Isopropanol Mix by Inversion Centrifuge Tubes at 3,000 rpm for 15 minutes Without Disturbing Pellet Pour Off Solution Add 5 mL 70% EtOH Centrifuge Tubes at 3,000 rpm for 10 minutes Set up Paper Towels for Drying Pour Off EtOH Set Tubes Upside Down on Paper Towels for 10 minutes Place Tubes Upright in Tube Racks Until Dry Add 500 µL LTE Store at Room Temperature Until End of the Day Centrifuge Tubes at 3,000 rpm for 5 minutes Label Eppendorf Tubes Transfer To Eppendorf Tubes Store at –20oC

Step 11

Step 22

Step 28

Summer Genomics Workshop Protocols

DNA Quantification (Gel Electrophoresis)
Equipment and Reagents Genomic DNA samples Lambda HindIII DNA Ladder Loading Dye Ice Bucket with ice Procedure 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 10. 11. 12. 13. Obtain Ice Bucket and Keep DNA Samples on Ice Heat HindIII Ladder at 60-65oC for 3 minutes Place on Ice Cut Small Parafilm Piece Pipet 3 µL dots loading dye onto parafilm Pipet 1 µL each DNA sample onto loading dye Pipet 1 µL HindIII DNA onto loading dye dot Record DNA positions in lab notebook Load gel Store remaining DNA at –20oC Plug gel box into power supply Set Voltage to approx. 80V Run gel until loading dye is approximately 1 1/2 – 2 inches below wells Visualize and photograph in gel doc room Dispose of gel Pipetman Pipet Tips Parafilm Permanent marker

Summer Genomics Workshop Protocols

DNA Quantification
Procedure

1. By comparing the brightness of the bands, you can estimate your DNA concentration. For instance, if your band is in halfway between the 4,361 bp and 2,322 bp fragments, you could estimate that your concentration is approximately 70 ng/ 1 g. 2. Alternately, you can determine the concentration by calculations. For example, if your band’s brightness seems similar to the brightness of the 4,361 band, you perform the following calculation 1 g/L 1 ng ------------- x 4,361 bp x ---------- = X ng/L 48,502 bp 1000 g

Summer Genomics Workshop Protocols

Exercises for Understanding
1. 2. What is a cationic detergent? Can you give another example other than CTAB? What does it mean that CTAB solubilizes membranes?

Summer Genomics Workshop Protocols


								
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