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Preparation of alpha-Factor DNA

Day 1
1. Digest a. b. c. d.

25g/l pDJ100 50l Buffer2 (NEB) 418.75l DEPC H20 6.25l Xba I (20,000 U/ml) ---------500l ---------minimum 2hrs @ 37C (can be o/n)

2. Run 4l on a gel next to 1l of uncut pDJ100 plasimd DNA to check for complete digestion 3. Add 495l of phenol:chloroform (1:1) to the reaction, vortex and spin for 15mins at RT in a microcentrifuge. 4. Transfer aqueous phase to a new tube 5. Add 495l of TE pH7.8 (10X TE= 100mM Tris-Cl pH7.8 plus 10mM EDTA pH8.0) to the organic phase, vortex and then combined the aqueous phase with the first one (~1ml) 6. Measure volume and split evenly between 2 tubes. Add 500l chloroform:isoamyl alcohol (24:1) to each tube, vortex and spin for 15min at RT in a microfuge. (* note: chloroform can be used instead of the chloroform:isoamyl alcohol solution) 7. Remove the aqueous phase to new tubes and measure volumes 8. Add 1/10th volume of 3M Sodium Acetate (pH 5.2) and 2X volume of 100% Ethanol. For example if tube1 contains 380l and tube2 contains 350l then: a. Tube1 = 380l b. 3M Sodium Acetate( pH 5.2) = 38l Tube2 = 350l 3M Sodium Acetate (pH5.2) = 35l

c. 100% Ethanol = 836l 9. Mix and place in the -20C freezer overnight

100% Ethanol = 770l

Day 2
1. 2. 3. 4. 5. 6. 7. Spin the DNA precipitate in a microfuge @ 14K, 4C for 15min Carefully remove the supernatant Fill each tube with 800l of ice cold 70% ethanol Spin for 2min in a microfuge @ 14K, 4C Remove supernatant with pulled pipet (do not aspirate) Air dry the pellet for ~5min @ room temp (ie. Leave tube top open) Add 41l of DEPC H2O to each tube, resuspend pellets and combine

Reagents for Transcription: 1. Make 1M DTT (154mg DTT in 1ml DEPC H2O) and aliquot. Freeze unused aliquots at -20C 2. Make up 10mM NTP: i. 6.0mg ATP ii. 5.9mg UTP iii. 5.8mg CTP iv. 5.9mg GTP add 1ml DEPC H2O and pH to 7.5 with 10M KOH (made in DEPC H2O) 1. (note: add only 1 to 5l of KOH at a time) aliquot and freeze unused aliquots at -20C 3. Make 5xNTP plus Capping Reagent: i. Add 300l DEPC H2O to a100l aliquot of 10mM NTP (resulting in a final concentration of 2.5mM NTP) ii. Add 216l of 2.5mM NTP to 5 Units of m7G(5)ppp(5)G (capping reagent P1-5-(7-Methyl)-guanosine-P3-5-guanosine triphosphate from Roche)

Transcription Reaction: Added to a 1.5ml Eppendorf tube in the following order: 20l 10X SP6 Buffer (from Roche) 47l DEPC H2O 1l 1M DTT 5l RNasin (from Promega) 40l 5x NTP with capping reagent 82l linerized pDJ100 5l SP6 Polymerase (20 U/l =100 U total; from Roche) ------200l ------Incubate at 37C for 1hr (use heat block) Extraction of RNA: Add 200l of phenol:chloroform (1:1), vortex and spin for 15min in a microfuge at 14K, 4C (note: It is important to perform the centrifugation to separate aqueous and organic phase in the cold 4 -10C. If performed at elevated temperatures, a residual amount of DNA (ie., linearized pDJ100) may sequester in the aqueous phase) Add 200l TE pH7.8 to phenol, vortex and spin

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