preparation of alpha-factor

7/31/2019 Preparation of Alpha-Factor

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Preparation of alpha-Factor DNA

Day 1

1. Digest

a. 25g/l pDJ100

b. 50l Buffer2 (NEB)

c. 418.75l DEPC H20

d. 6.25l Xba I (20,000 U/ml)

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500l

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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 Tube2 = 350l

b. 3M Sodium Acetate( pH 5.2) = 38l 3M Sodium Acetate (pH5.2) = 35l


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c. 100% Ethanol = 836l 100% Ethanol = 770l

9. Mix and place in the -20C freezer overnight

Day 2

1. Spin the DNA precipitate in a microfuge @ 14K, 4C for 15min

2. Carefully remove the supernatant

3. Fill each tube with 800l of ice cold 70% ethanol

4. Spin for 2min in a microfuge @ 14K, 4C

5. Remove supernatant with pulled pipet (do not aspirate)6. Air dry the pellet for ~5min @ room temp (ie. Leave tube top open)

7. 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 H 2O)

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)


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Transcription Reaction:

Added to a 1.5ml Eppendorf tube in the following order:

20l 10X SP6 Buffer (from Roche)

47l DEPC H2O

1l 1M DTT5l RNasin (from Promega)

40l 5x NTP with capping reagent

82l linerized pDJ100

5l SP6 Polymerase (20 U/l =100 U total; from Roche)

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200l

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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, 4 C

(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

preparation of alpha-factor

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