Lab Protocols
Agarose Gel Electrophoresis
1. Make up sufficient agarose for the electrophoresis unit to be used. The small gels require ~50 mls the large gels require ~300 mls. (If cutting out a band for cloning use LMT (Low Melting Temperature) agarose and TAE buffer
| 100mls 0.75% | 100 mls 2% | 300 mls 0.75% |
| 5 ml 10X TTE | 5 ml 10X TTE | 15 ml 10X TTE |
| 0.75 g agarose | 2.0 g agarose | 2.25 g agarose |
| 90 ml H2O | 90 ml H2O | 270 ml H2O |
2. Cover flask with plastic wrap and microwave on high for 4 minutes. Use a hot mitt to handle flask. Carefully swirl and be sure all agarose is dissolved. If not, microwave one minute at a time until all is thoroughly dissolved.
3. Let agarose cool until you can hold your hand on the flask for three seconds. Ethiduim Bromide is a 10 mg/ml stock, which is 20,000X. Add EthBr carefully-it is a carcinogin. (100 ml/20,000 = 5
ml)4. Pour agarose into taped gel caster, filling it 2/3 full. Then put the comb in place, being sure that the comb is parallel to the top egde. Let harden. Meanwhile, make up enough 1X TTE buffer to fill the electrophoresis chamber. (Small ~250 ml, large ~ 1 L)
5. When agarose is hard, carefully remove the comb from the gel. Remove the tape from the ends, being carefully that the gel does not slide away. Add 1X TTE buffer to the apparatus so that the buffer is above the gel. Add 10X DNA loading buffer to sample so that the final concentration is 1X, and load samples into wells. Remeber to include a size standard.
6. Run at ~50V- Remember, "run into the red"
10X DNA loading buffer
for 10 ml
40% sucrose 4 g
250mM EDTA 5 ml of 500mM stock
H2O fill to 10 ml
~1.5% Brompheno blue tap in powder "to taste"
Competent bacteria: CaCl2 method
ml each into sterile eppendorf tubes and store in the -80° C freezer. KEEP CELLS COLD WHILE ALIQUOTING.Grow a 5 ml overnight culture of the desired bacterial strain
Make a 1:100 inoculation into 200 ml LB in a 500 ml flask.
Grow to an OD600 of 0.4 (up to 0.45)
Chill on ice for 15 min.
Spin 10’, 5000 rpm, 4° C in the GSA rotor
Resuspend in 1/4 original volume (50 ml) of cold 0.1 M CaCl2
Incubate on ice for 20’
Spin again, 10’, 5000 rpm, 4° C in the GSA rotor
Resuspend in 1/20 original volume (5 ml) of cold 0.1 M CaCl2
Leave on ice in covered ice bucket in cold room over night
Add sterile glycerol to 10% (1 ml of 50% glycerol)
Make aliquots of 200
ml aliquot and divide into two aliquots of 100 ml each (be sure the new tube is pre-chilled on ice)Thaw a 200
Add DNA to be transformed. Generally, add 1
ml of supercoiled plasmid DNA OR 10 ml of a ligation reaction. Be sure to include a control tube that does not get any DNA.Incubate on ice for 5’
Heat shock the cells for 45’’ at 42°C.
Return the tubes to ice
Add 1 ml of LB broth to each tube, mix well
Incubate for ~ 30’ at 37° C
Plate onto the appropriate selective media.
ml and the rest of the mediaSuggested plating amounts:
Ligation reactions: 2 plates, 100
Plasmid DNA: 2 plates, 50
ml and 250 mlNOTE: If the frozen cells warm up (in the event of a freezer crash or power outage) the cells will lose their competency and should be tossed.
Commonly used strains
Name |
Genotype |
| DH5a (JKe285) | supE44 DlacU169(f80lacDZM15) hsdR17 recA1 gyrA96 thi-1 relA1 |
| JBe15 | pyrF(ura) trpC ampS kanR |
| MC1666 (JBe181) | pyrF(ura) trpC LeuB (leu1) |
| TG-1 (fast growth, nucleases active ) | supE D(lac-proAB) hsdD5 F’ traD36 proAB+ lacIq lacZ DM15 |
Competent Bacteria-TSB cells
Grow a 5 ml overnight culture of the desired bacterial strain
Make a 1:100 inoculation into LB (100 ml)
Grow to an OD600 of 0.3-0.6*
Chill on ice for 15 min.
Centrifuge 1,000xg (5.5K) for 10 minutes at 4° C
Resuspend in 1/10 original volume (10 ml) transformation and storage buffer (TSB) at 4C and incubate on ice for 10 minutes
Cells can be used immediately or frozen in liquid nitrogen or dry ice/ethanol bath and stored at –70C.
Transform as for the CaCl2 protocol (see page 3). It is recommended that after heat shock, TSB with 20mM glucose.
TG-1 recommended OD600 of 0.2-0.3
DH5a recommended OD600 of 0.4-0.5
TSB
LB broth with Add the following to 100 ml of LB broth:
10% PEG 10 g PEG (MW 3,350)
5% DMSO 5 ml DMSO (wear gloves-use caution)
20mM MgCl2 2 ml 1 M MgCl2
Filter sterilize or autoclave.
Yeast Genomic DNA Preparation
Glass beads method
(Volumes and weights given are for 10 ml cultures)
1. Grow ~2 x 108 cells (OD660=1.0; Klett=50-100; 1-2 x 107 cells/ml)
[inoculate 10 ml with matchhead size cell clump and grow overnight]
Pellet in tabletop centrifuge: 1500 rpm, 5 minutes
2. Resuspend pellet in 1 ml H2O, mixing well, and transfer to a screw cap eppendorf tube.
3. Quick spin to pellet cells and resuspend in 1 ml H2O, mixing well.
4. Quick spin to pellet cells and resuspend in 0.2 ml extraction buffer
Freeze/thaw cells (-75°C) to help lysis.
Extraction buffer:
for 100 mls
0.5 M NaCl 10 ml 5 M NaCl
0.2 M Tris-HCl, pH 7.6 10 ml 2 M Tris
0.01 M EDTA 2 ml 0.5 M EDTA
+/- 1% SDS 10 ml 10% SDS
5. Add glass beads (0.45-0.5
mm) to the 0.4 ml-0.5 ml mark.6. Add 0.2 ml PCIA [BOTTOM LAYER!] (PCIA is an equal volume of phenol and chloroform/isoamyl alcohol that has been equilibrated with a volume of extraction buffer [minus SDS] equal to the volume of phenol/chloroform)
7. Vortex for 2.5 minutes
8. Add 0.3 ml buffer (no SDS) and 0.3 ml PCIA. Vortex 1 minute. Spin in microfuge 4 minutes.
9. Transfer 400
ml of aqueous phase (upper) to a fresh tube. (Discard the beads and organic phase in the hood.) Add 800 ml of ethanol to the aqueous phase and mix well by inverting. Incubate on ice for 10 minutes. Spin 10 minutes in microfuge.10. Wash pellet with 70% ethanol, aspirate, and air dry.
11. Resuspend in 50
ml TE. Use 5 ml per 20 ml digest:stock for 10 reactions:
5
ml DNA2
ml 10X buffer 20 ml 10X buffer1
ml enzyme ___ ml enzyme1
m RNAase 10 m RNAase11
ml H20 ____ ml H20 to a total volume of 450 mlDigest several hours ---> overnight.
Yeast Genomic DNA
Qiagen tissue kit method
Sorbitol buffer: for 10 ml
1 M Sorbitol 1.82 g
100 mM EDTA 2 ml 0.5 M
14 mM b-mercaptoethanol 100 ml
Grow 5 ml yeast culture in YPD overnight at 30C (recommended to OD600 of 10)
Place 1.5 ml in a microfuge tube. Spin to pellet
Resuspend pellet in 600 ml sorbitol buffer. Add 200U of zymolyase or lyticase and incubate at 30C for 30 minutes
Pellet spheroblasts by centrifuging for 5 minutes at 7500 rpm (5000X g)
Resuspend the spheroplasts in 180 ml of buffer ATL supplied in Qiagen kit.
Follow the QIAmp tissue protocol from step 2 as follows:
add 20 ul of Proteinase K stock solution, mix by vortexing, and incubate at 55 C for 4 hours-overnight. Vortex occasionally during incubation.
Centrifuge for 5 min at full speed. Pipet 200
ml to a fresh microfuge tube.Add 410 ul of the Buffer AL/ethanol mixture to the sample, and mix thoroughly by vortexing.
place a QI amp spin column in a 2 ml collection tube. Carefully apply the mixture from step 4 (including the precipitate) to the QI amp spin column without moistening the rim, close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min.
place QI amp spin column in a clean 2 ml collection tube, and discard the tube containing the filtrate.
Carefully open QI amp spin column and add 500 ul of Buffer AW. Centrifuge at 6000 x g for 1 min.
Place QI amp spin column in a clean 2 ml collection microtube, and discard the collection tube containing the filtrate. Open QI amp spin column and add another 500 ul of Buffer AW. Centrifuge at 6000 x g for 1 min, and at full speed for an additional 2 min.
place QI amp spin column in a clean 1.5 ml microfuge tube, and discard the collection tube containing the filtrate.
open the QI amp spin column. Elute the DNA twice by adding 200 ul of Buffer AE or distilled water preheated to 70 C, incubate 1 min. at room temperature, then centrifuge 1 minute at 6000xg. I flots of DNA is needed, repeat the elution with another 200 ml into a fresh 1.5
ml microfuge tube and pool the eluates into a single tube.
Ethanol Precipitation of DNA or RNA
Ethanol precipitation of nucleic acids is done either to concentrate the DNA or to remove impurities.
1. Using a 5 M NaCl stock solution, adjust the salt concentration of the DNA solution so that it is betweem 250 mM and 500 mM. Be sure to consider salts that may already be present, such as the salt in restriction enzyme buffers.
2. Add 2-2.5x the volume of 95% cold ethanol. Immediately mix well by inverting the tube several times. (A 1x volume of isopropanol can also be used.)
3. Incubate in an ice bucket for 20 minutes.
4. Spin in the microfuge for 15 minutes. Make sure that the hinge of the microcentrifuge tube is pointed out so you know where your pellet is. If precipitating a large volume, spin in a corex tube in the Sorvall superspeed at 10,000 rpm for 10 minutes. Be sure to mark the side of the tube where the pellet will be.
5. Aspirate the ethanol, rinse the pellet by squirting a small amount of 70% ethanol down the side of the tube, and then aspirate again. Allow the pellet to dry a few minutes, and then resuspend in an appropriate volume of TE.
Washing Glass Beads
Rinse beads with 15 changes of DI water. Be sure each rinse is thorough by stirring beads vigorously with a spatula.Rinse beads 3 times with 50% ethanol
Decant as much liquid as possible. Remove excess liquid by placing aliquots of beads in a buchner funnel with teflon filter material (funnel and teflon circles are located in a metal pan under the small vacuum pump in the micro lab)
Dump vacuumed beads into the metal pan. Bake in 180°C oven until thoroughly dry (several hours). Pour into glass beakers and cover with foil. Put in cold room.
Kinasing Oligonucleotides
g-32P-ATP (3000 Ci/mmol) 5 ml (~117 pmol)oligo 2 ml (100 pmol)
10X kinase buffer 1 ml
kinase 1 ml
H2O 1 ml
Incubate 60 minutes, 37°C
Purify oligo from unincorporated nucleotides on a G25 spin column
(Instructions for preparing G25 are in Maniatis)
1. Add well mixed resin to top of skinny part of column (3.5 ml)
2. Spin 5’, 1000 rpm in 15 ml conical
3. Empty liquid from conical and place catch tube in bottom
4. Add 90 ml TE to kinase reaction and load all 100 ml onto column
5. Spin 2’, 1000 rpm
6. Add 300 ml TE to column (don’t remove from centrifuge to do this)7. Spin 5’, 1000 rpm
8. Place column in radioactive waste. Recover catch tube containing kinased oligo
Count 1 ml (multiply x2 for dry counting)
add to blot at ~0.5-1x106 cpm/ml ( at least 1 pmol/ml hyb solution)
Add the following to a microfuge tube:
0.1– 0.25 ml probe
200 ml carrier DNA
700 ml hybridization solution.
Heat 2 minutes, 100C, add to rest of hyb solution in a conicol tube. Mix well and add to blot.
LIGATIONS
The key to ligations is getting the vector:insert molar ratio somewhere close to 1:1. This is difficult because the DNA concentration is often difficult to determine accurately. Thus, it is best to set up several reactions using a range of vector concentrations.
1. Sample set-up:
| reaction 1 | reaction 2 | reaction 3 | reaction 4 | |
| H2O | 17 ml | 14 ml | 14 ml | 14 ml |
| 10X ligase buffer | 2 ml | 2 ml | 2 ml | 2 ml |
| vector | 1 ml | 1 ml | 1 ml @ 1:10 | 1 ml @ 1:00 |
| insert | 0 ml | 3 ml | 3 ml | 3 ml |
| ligase enzyme | 0.5 ml | 0.5 ml | 0.5 ml | 0.5 ml |
2. Incubate ligation reactions at 22ºC for several hours or overnight.
NOTES:
A. Ligase enzyme:
ALWAYS use a fresh, sterile tip
keep on ice at ALL times
handle tubes at the tops ONLY
pipette carefully and accurately-place the pipet tip on the surface of the enzyme solution. BE VERY CAREFUL not to immerse the tip-the glycerol sticks to the outside of the tip resulting in inaccurate pipeting and wasted enzyme.
B. DNA in LMT agarose:
Heat the gel slice for 5 minutes at 68ºC, and add to tube containing waterand buffer. Be sure that the liquid in the tube has cooled to 37°C before adding ligase enzyme (shouldn’t feel hot), but don’t wait too long or it will gel and not mix properly. The ligation and transformation reactions are inhibited when more than 3
ml of LMT gel is added to a 20 ml reaction.C. For transformation, add 10
ml of the ligation reaction to 100 ml of competent cells . If the ligation reaction has LMT agarose in it, heat to 65ºC for 3 minutes and cool slightly before adding.
Standard PCR reaction
per reaction: final concentration
22.5 ml H2O
5 ml 10x PCR buffer 1x
10 ml dNTP mix (1 mM ea. dNTP) 200 mM
0.5 ml primer 1, 20 mM 0.2 mM
0.5 ml primer 2, 20 mM 0.2 mM
10 ml DNA 10 ng (plasmid DNA)
0.5 ml Taq polymerase, (5 U/ ml) 2.5 U/100 ml
1 minute 94C
1 minute anneal (5-10 degrees below primer annealing temperature)
2 minutes 72C (~1 min extension/kb of DNA product)
25 cycles
10X PCR buffer: for 1 ml:
500 mM KCl 166.7 ul of 3M stock
100 mM TRIS/HCl pH8.3 50 ul of 2M stock
15 mM MgCl2 15 ul of 1M stock
768.3 ul water
* Note: STD condition is 1.5mM MgCl2 Optimum usually somewhere in the range of 1.5-4 mM (empirically determined). Some protocols also include 0.1% gelatin or BSA.
dNTP mix (1.0 mM ea. dNTP):
5 ml 100mM dATP
5 ml 100mM dCTP
5 ml 100mM dGTP
5 ml 100mM dTTP
480 ml H2O
25 mM MgCl2
Control reaction conditions are given at the end of the Taq purification protocol.
PCR RT assay
This protocol is a revision of the assay described in:
Pyra, H, Böni, J. and Schüpbach, J. Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement. PNAS 91, 1544-1548. (Feb. ‘94)
1. Prepare primer/template
times
per reaction: # reactions = total
mlMS2 RNA (0.8 mg/
ml) 0.5 ml[~0.28 pmol]
primer RT-1 (9 pmol/ml) 1 ml
[~72 ng]
Incubate at 95ºC, 5 min; 37ºC, 20 min.; then on ice 5 min.
2. Prepare source of RT
While primer/template is incubating, label PCR tubes and put a0.5 ml of a 1:10 dilution of each VLP sucrose gradient fraction to be tested per tube. Be sure to include a positive control with 0.5 ml of purified RT and a negative control with 0.5 ml of 30% sucrose.
3. RT reaction
While primer/template reaction is on final ice incubation, prepare a stock of the RT reaction mix.
times
per reaction: # of reactions = total
ml5x endogenous RT buffer 5
mlH2O 16
mlRNase inhibitor (20 U/ul) 0.5
mlwater 2.0
ml ______Combine this with the primer/template, then add 23.5 ml to each PCR tube containing the VLP fractions. Incubate at 22ºC for 1 hour.
4. PCR reaction
Incubate RT reactions at 95ºC for 5 minutes to inactivate the RNase inhibitor. During this incubation, make up the PCR mix:
times
per reaction: # of reactions=total
ml5x PCR mix 15
mlwater 60
mlTaq (5U/
ml) 0.5 ml
Quick spin tubes after 95°C incubation, and add 75 ml PCR reaction mix.
Cover reaction with three drops of mineral oil.
Incubate at 37ºC for 15 minutes to digest the RNA template.
PCR for 25 cycles
94ºC, 1 minute
55ºC, 1 minute
72ºC, 30 seconds
Run 50 ml of reaction on an 8% PAG or 3.0% agarose gel.
Amplification produces a 112 bp fragment representing positions 21-132 of the 5’ end of the MS2-RNA (GenBank accession #J02467)
If desired, electroblot gel to membrane and probe with oligo RT-3.
REAGENTS AND BUFFERS
5x Endo mix (not -G!) for 1 ml
50 mM MgCl2 50 ml 1 M MgCl2
250 mM TRIS-HCl (pH 8.0) 125 ml 2M TRIS
2% b-ME 100 ml
500 mM each dNTP 50 ml 10mM dATP
50 ml 10mM dCTP
50 ml 10mM dGTP
50 ml 10mM dTTP
475 ml H2O
5X PCR mix for 1.5 ml for 1.0 ml
primer RT-1 (@ 90 pmol/ml) 15 ml 10 ml
primer RT-2 (@ 25 pmol/ ml) 10 ml 6.67 ml
RNase (@ 8 ng/ ml) 100 ml 66.7 ml
3M KCl 125 ml 83.3 ml
2M TRIS/HCl, pH 8.3 37.5 ml 25 ml
100 mM dATP 10 ml 6.67 ml
100 mM dCTP 10 ml 6.67 ml
100 mM dGTP 10 ml 6.67 ml
100 mM dTTP 10 ml 6.67 ml
water 1172.5 ml 782 ml
Primer RT-1: 5’-CATAGGTCAAACCTCCTAGGAATG-3’
Primer RT-2: 5’-TCCTGCTCAACTTCCTGTCGAG-3’
Primer RT-3: 5’-TTAATGTCTTTAGCGAGACGC-3’
Bacteriophage MS2 RNA Boehringer Mannh eim #165 948
RNase inhibitor, 2000U Boehringer Mannheim # 799 017
Phenol/Chloroform extraction
Phenol and chloroform are nasty chemicals. Wear gloves, and please, no inhaling!
INTRODUCTION
Phenol/chloroform extraction is done to remove proteins (cellular or added enzymes) from a DNA prep. The phenol denatures the proteins, and after centrifugation, the proteins lie in the interface between the organic and aqueous phases.
Phenol is melted with TRIS buffer, mixed 5:1 with chloroform, and kept in a brown bottle to protect it from the light. The organic phenol phase is always on the bottom. When pipeting, be sure to get the lower phase. Pipet carefully, putting the pipet or pipetman tip through the aqueouse phase. Be sure not to get the pipetman itself down into the aqueous phase-it will contaminate it.
Chloroform is kept in a glass stoppered bottle, saturated with water and iso-amyl alcolhol. The chloroform is always the bottom phase. Pipet carefully, being sure that your pipetman does not contact the upper aqeoues phase.
PROCEDURE
1. Add a volume of phenol so that the total volume is equal to the DNA volume (e.g. if your DNA prep is in 100ul, add 100ul phenol). Vortex for several seconds. Spin for 5 minutes (microfuge or table top at 2000 rpm depending on volume)
NOTE: After centrifugation, you should see two distinct phases. The upper is the aqueous, the lower is the organic. The upper aqueous phase may be cloudy-that’s O.K.
2. Carefully transfer the upper aqueous phase to a fresh tube, being careful not to transfer any of the interface material.
3. Add an equal volume of chloroform, vortex well, and centrifuge as above.
4. Carefully transfer the upper aqueous phase to a fresh tube. The DNA should now be ethanol precipitated to remove any remaining organics.
Yeast Plasmid Rescue
Streak cells from a fresh plate onto 1/6th of an SC plate keeping selection for the plasmid. Grow overnight at 30C
Scrape cells from plate with a sealed capillary tube into 0.1 ml STET buffer
(Alternatively, prepare 5mL o/n culture of yeast cells Pellet cells and resuspend in 0.1mL STET buffer, transfer to microfuge tube.)
Add 0.2g glass beads (slightly over top of liquid), vortex for 5 min @ 4o C.
Add 0.1mL STET buffer, boil for 3 min.
Cool briefly on ice, microfuge for 10 min at 4o C.
Transfer 0.1 mL of supernatant to 50mL of 7.5 M ammonium acetate. Incubate at 4o C for 1 hour, microfuge for 10 min at 4o C.
Add 0.1mL of supernatant to 0.2mL ice-cold EtOH, microfuge as above.
Decant pellet, wash with 70% EtOH. Dry, then resuspend in 20mL H2O.
Use 10mL of suspension for transformations into E. coli. Use JBe181 if need to be able to select for URA or LEU plasmids.
Total Protein Preparation from Yeast (denatured)
Steps 1-6, keep on ice or do in the cold room (4ºC)
1. spin down cells from 5 ml culture (5’, 2000rpm table top) at ~2x107 cells/ml
2. wash 1x with H2O by resuspending in 500 ml H2O and transferring to an eppendorf tube. Pellet cells with a 10 second spin and remove supernatant.
3. resuspend in 500 ul of Buffer B+ 5mM DTT + 2mM PMSF. Quick spin and remove supernatant.
4. resuspend in 0.4 ml Buffer B+ 5mM DTT + 2mM PMSF.
5. add glass beads to meniscus, vortex at top speed for 5 minutes. Put tubes back on ice immediately.
6. add 0.1 ml buffer B+5 mM DTT + 2 mM PMSF, vortex, take supernatant (~250 ml). Go down into beads with yellow tip and get all liquid possible.
7. add equal volume of 2X sample buffer, mix well
8. boil immediately for 5 minutes
9. spin 15 minutes , transfer 500 ml supernatent to a fresh tube, store at -20ºC
For SDS-PAGE, run 15-20 ml/lane
For TyB use 25 ml
Buffer B: for 50 ml
15 mM KCl 0.75 ml 1 M KCl
10 mM HEPES/KOH pH7.8 1.0 ml 0.5 M HEPES/KOH
5 mM EDTA 0.5 ml 0.5 M EDTA
2X sample buffer: Buffer B + 5mM DTT+ 2 mM PMSF:
2.0 ml glycerol 10 m l PMSF (100mM)
1.0 ml 2-ME 25 m l DTT (1M)
0.625 ml 2M TRIS, pH 6.8 5 ml Buffer
1.375 ml H2O
5.0 ml 10% SDS
a pinch of bromphenol blue
PROTEIN GELS
RUNNING GEL (7.5%):
RUNNING GEL BUFFER (1.5M):3.77 ml of 40% acrylamide & Bis (37.5:1) 36.3 g Tris (MW=121.14 g/mol
5.0 ml Running Buffer Add ~ 70% of total volume
0.2 ml (or 200 ul) of 10% SDS Adjust to pH 8.8 with HCl
11.0 ml H2O (milli-Q) Add H2O (milli-Q) to 200 mls
100 m l 10% AP (ammonium persulfate)
10 m l TEMED
20 ml total
STACKING GEL(4%): STACKING GEL BUFFER (0.5M):
1.O0 ml 40% acrylamide & Bis (37.5:1) 3.03 g Tris
2.5 ml Stacking Buffer Add ~50-60% of total volume
100 m l 10% SDS Adjust to pH 6.8 with HCl
6.3 ml H2O (milli-Q) Add H2O (milli-Q) to 50 mls
50 m l 10% AP (ammonium persulfate)
4 m l TEMED
10 ml total
TANK BUFFER (10X): RUNNING GEL(10%):
33 g Tris 5.0 ml of 40% acrylamide & Bis
144 g glycine 5.0 ml Running Buffer
10 g of SDS 0.2 ml (or 200 ul) of 10% SD
Use magnetic stir bar & add H2O to 1 liter 9.7 ml H2O (milli-Q)
100 m l 10% AP
10 m l TEMED
20 ml total
PROTOCOL: GEL SET-UP
1. Clean plates well with soap & rinse with DI water . Rinse sides of plates that will be in contact with gel using 70% ethanol. Clean & rinse side spacers and comb with ethanol and water. Put glass plates together, ensuring that the side spacers and bottom spacer are firmly attached, use 6 clips to combine plates.
2. Pour Running Gel (using plastic pipettes). Make sure to hold pipette in a corner ( do not stop pouring and restart because this will create bubbles in the gel). Pour gel until there is a space below where the combs will be that is approximately the height of the "teeth". Add a thin layer of isobutanol on top of gel (running gel won’t polymerize in prescence of oxygen). Let this polymerize before pouring the stacker (~40 min., or until a shadow can be seen behind the line where the gel stops) . Once polymerized, pour off isobutanol and wash with water (milli-Q) many times. Make sure that all of the water has been removed from between the plates ( use a paper towel as a wick to draw out the water).
3. Pour stacker gel on top of polymerized running gel, put comb in, and be careful not to create any bubbles while doing so. Let this polymerize. Shadows will appear around the teeth when the gel is polymerized.
LOADING GEL:
1. Put gel into electrophoresis chamber and add buffer to lower container. Make sure the bottom spacer is removed. Use a "bent" syringe to flush out the bubbles at the bottom of the gel. In order to do this, one must ensure that the syringe is between the plates.
2. Next, add buffer to the upper chamber. Make sure that the buffer rises above the comb. Get a straight syringe and pull out the comb. Immediately use the syringe to flush out unpolymerized acrylamide that is in the wells.
3. Now load the protein marker and samples. (example fpr techniques class)
Lane 1: marker: 10 m l
Lane 2: undiluted protein (from JKc608) in glucose: 20 m l
Lane 3: 1:2.5 dilution of protein in glucose: 20 m l
Lane 4: undiluted protein in galactose: 20 m l
Lane 5: 1:2.5 dilution of protein in galactose: 20 m l
Lane 6: marker: 10 m l
Lane 7: undiluted protein (from JKc608) in glucose: 20 m l
Lane 8: 1:2.5 dilution of protein in glucose: 20 m l
Lane 9: 1:5 dilution of protein in glucose: 20 m l
Lane 10: undiluted protein in galactose: 20 m l
Lane 11: 1:2.5 dilution of protein in galactose: 20 m l
Lane 12: 1:5 dilution of protein in galactose: 20 m l
4. Run gel at ~38 mAmps for ~4 hours or until marker gets to the very bottom of the gel. Do not run marker off the gel.
Note: Heat markers and samples for 3 min. @100oC to denature proteins before loading on gel.
SAMPLE DILUTIONS:
1:2.5 1:5
40 m l sample protein 20 m l sample protein
30 m l 2X buffer 40 m l 2X buffer
30 m l water 40 m l water
100 m l total 100 m l total
PROTEIN GEL STAINING
Place gel containing lanes 1-5 in a sol’n of Commassie Blue stain (use a glass dish)
Let stain for an hour
Drain stain back into bottle for reuse. Rinse off remaining stain with Destain.
Heat Destain in microwave until warm to touch (~ 1 minute)
Pour Destain in dish with gel, add a few layer of kim wipes (cut to gel size) and then add a few pieces of 3MM filter paper, put saran wrap on top of dish and let set overnight.
Dry gel next day. (see instruction manual to gel dryer)
Coomassie Blue R
45% methanol
9% acetic acid
0.25% (weight) Coomaise
Protein gel transfer for Western Blotting
1. Run acrylamide gel to separate proteins. For Ty1 proteins, the recommended is 7.5% gel for TyB and 10% for TyA. The thin gels (.8mm) can hold about 20ml. per lane maximum.
2. For transfer, wet nitrocellulose in water; wet immobilon-P (or similar membranes) with 100% CH3OH and rinse with H2O. Soak the gel and the transfer membrane in the transfer buffer (1X) for 10 minutes.
Transfer Buffer (10X) Transfer Buffer (1X)
15.1g Tris 100ml 10X transfer buffer
72g glycine 100ml 10% methanol
add H2O to 1 L add H2O to 1L
** never adjust the pH of an electroblotting buffer with HCl. The Cl that will be generated will severely corrode the electrode connections
Note: The methanol in the transfer buffer is for fixing of proteins to the membrane, and is recommended for transfer of small proteins. Some claim it may impede transfer of large proteins by fixing them in the gel, but we have not seen this problem. Some people include SDS in their transfer buffer. SDS is supposed to help transfer large proteins, but increases chances of bubbles in the transfer. In a side by side comparison it did not improve transfer of a 180Kd protein and is not recommended.
3. Proper care of western blotting electrodes
a. The platinum electrode plate should be used as the + electrode only. DO NOT SCRUB these electrode plates. It is marked plattinum, and the electrode connection is on the upper right.
b. The alloy plates are to be used as the - electrode. They may be used as the + electrode, but this is not recommended as discoloring and pitting will occur, especially in transfers longer than two hours.
c. Do not do overnight transfers, as it causes unnecessary breakdown of the electrodes.
4. Transfer set-up:
Note: For efficient transfer, it is extremely important to get rid of all of the bubbles and to pack the transfer set up snuggly.
Put items into the plastic holding tray in the following order:
- electrode
plastic rack
1-2 sponges (or more if thin)
one piece of 3MM blotting paper cut to size (roll out bubbles with test tube)
the protein gel
the transfer membrane
one piece of 3MM blotting paper
carefully add more buffer
1-2 sponges (or more if needed)
plastic rack
connect a red lead wire to the + electrode and place it carefully on top
carefully fill tray with buffer
feed the red lead through the right hand hole of the plastic cover and place cover on top
contact the black lead wire to the - electrode on the bottom
The height of the set up sould be ~2 mm higher than the plastic tray
Place the entire holder with set-up at the edge of the sink and ease it into the stand, and slowly turn upright - hold over sink so overflow goes into the sink! Place in plastic tray for transfer to collect overflow.
5. Transfer:
24V for 60 minutes transfers TyA /TyB (180Kd) proteins
General guidelines: most western blots of 1mm gels are complete in 30 minutes, 6mm gels in two hours. The field strength of the blotter at 24V is approximately that of our protein gel boxes run at 240V (12V/cm). Thus the time required for blotting will be about the same amount of time it takes a band of molecules to move a distance equal to the thickness of the gel down the gel. 24 V is the maximum recommended voltage level.
Electrotransfer of Nucleic Acids from Acrylamide Gels
For DNA, use unpHed TBE
89mM Tis-Borate, 2 mM EDTA (pH will be approximately 8.0)
Tris base 10.8 g
Boric Acid 5.5 g
EDTA 0.76g
Dilute to 1 liter with water and use at 0.5X
Transfer at 12V for 20 minutes with a 1mm gel thickness
For RNA use MOPS buffer (pH 7.0)
20 mM MOPS
5 mM Sodium Acetate
0.5 mM EDTA
For 500 ml of 10X:
23.1 g MOPS
3.4 g Sodium Acetate
0.93 g EDTA
adjust to pH 7.0 with acetic acid or NaOH
Transfer at 6V for 20-30 minutes.
PROTEIN HYBRIDIZATION AND DETECTION WITH ECL:
1.
After transfer, mark protein side of nitrocellulose. Visually check transfer by staining membrane in PonceauS for ~5 minutes. Return PonceauS to bottle. Rinse off blot with stream from water bottle. Mark positions of standards.2. Block 30 min. @ room temperature (use rocker) or overnight at 4o C in 5% dried milk, PBS-T (phosphate buffered saline with .05% Tween-20)
3. Wash 2X 5 min. with PBS
4. Hybridize primary Ab, 1 hour (Ab diluted in PBS)
5. Wash 2X 5 min. with PBS
6. Hybridize secondary Ab (anti-primary, IgG, HRP conjugated) for 30-60 min. @ Rm. temp.
7. Wash 1X 15 min. in PBS, and 4X 5 min. in PBS-T
8. Mix developing reagents 1 and 2 at 1:1 ratio with a 10 ml total. The reagents only last ~ one hour. Pour mixed reagents onto an old film and lay protein side of nitrocellulose down in reagent for one minute. Blot off excess reagents with Kim wipes and wrap nitrocellulose in saran wrap.
9. Immediately place nitrocellulose in cassette and take down to the dark room.
10. Put safe light on and remove film from box, put into cassette.
11. Exposure times can vary, develop short exposures and see if a longer one is needed.
12. After exposure, remove film and put in developer solution for 3 min.
13. Rinse film in water for ~30 seconds
14. Put film in fixer solution for 5 minutes
15. Rinse film with water for 5 minutes.
16. Air dry film.
5% dried milk, PBS-T(.05% Tween-20) PBS:
5g dried milk 4.62g FTA hemagluttinin buffer
50 m l Tween-20 add to 500 mls with milli-Q H20
add PBS to 100 mls
PBS-T:
100 m l Tween-20
add PBS to 200 mls
Ty antibody information:
For Ty1 TyA protein use R1 rabbit polyclonal antibody
R1-F is used at 1:2500 (20
ml in 50 ml)R2-F is used at 1:10,000 (5
ml in 50 ml)Secondary antibody is goat anti-rabbit IgG-HRP conjugated at 1:16,000
For Ty1 TyB protein use mouse monoclonal 8B-11 (to integrase) column purified 1:1000
Secondary antibody is goat anti-mouse IgG-HRP conjugated at 1:18,000
For Ty3 capsid use anti-capsid (26 kD) IgG purified rabbit polyclonal at 1:000
I. O.D. for quantification of DNA and RNA
Make a 1/100 dilution of sample in water. 10 ml in 990 ml is recommended.
Measure the O.D. at 260 nm and at 280 nm. (Remember to pull out the black lever, flip the switch and push the button to turn the U.V. lamp on.) Must be measured in quartz cuvettes.
The ratio of readings gives an estimate of purity.
Pure DNA: OD260/OD280 has a value of 1.8
Pure RNA: OD260/OD280 has a value of 2.0
Contamination with protein or phenol decreases these ratios, and quantitation by the numbers will not be accurate.
The OD260 allows quantitation of nucleic acid.
An OD of 1 corresponds to the following:
50 mg/ml for double-stranded DNA
40 mg/ml for single-stranded DNA and RNA
20 mg/ml for single-stranded oligonucleotides
II. ESTIMATING DNA CONCENTRATION OF UNCUT PLASMID
1. Melt agarose of 0.75-1.0% agarose (with EtBr). Pour molten agarose slowing onto a microscope slide until it just covers the surface. Allow to harden.
2. Spot 1 ul each of a series of lambda DNA dilutions from 0.5 ug/ul. (Dilution series is in the DNA markers box. Spot 1 ul of DNA sample near-by.
3. Observe by U.V. light to find nearest concentration
Restriction Enzyme (R.E.) digests
Enzymes are expensive-
ALWAYS use a fresh, sterile tip when pipetting enzymes!
keep on ice at ALL times
handle tubes at the tops ONLY
pipette carefully and accurately-place the pipet tip on the surface of the enzyme solution. BE VERY CAREFUL not to immerse the tip-the glycerol sticks to the outside of the tip resulting in innaccurate pipeting and wasted enzyme.
check the incubation temperature in the catalog
Enzymes are shipped in 50% glycerol. The concentration is written on the label in units. One unit of enzyme is defined as digesting 1 ug of DNA in 1 hour at the optimum temperature. Most enzymes are about 10,000U/ml (10U/ul). Enzymes are diluted prior to use. The total concentration of glycerol should not be higher than 5% (1/10th of the total digest volume). Each enzyme has its own preferred buffer, which is listed in the New England Biolabs (NEB) catalog. A vial of 10x concentrated buffer is sent with each enzyme. ALWAYS use a fresh, sterile tip when pipetting enzymes!
A typical enzyme digest:
16 ul H2O (add first)
2 ul 10X bufffer (use same tip as used for water)
2 ul DNA (use fresh tip)
0.5 ul enzyme (use fresh tip)
20 ul
When digesting multiple DNAs with the same enzyme, make a stock solution. Multiply each of the component volumes by the number of samples +1, mix well, and aliquot to tubes containing DNA to be digested.
RNA Extraction from Yeast
1. Spin down 1 ml of saturated liquid culture (10 mls if minimal or drop-out media) in table top for 5’ at 2000 rpm
2. Resuspend in 0.3 ml RNA buffer, chill cells on ice
3. Add 0.4 g glass beads
0.15 ml phenol
0.15 ml chloroform/isoamyloh
4. Vortex at top speed, 30 seconds
5. Spin in microfuge in cold room, 5 minutes
6. Remove 250 ml aqueous phase to fresh tube, add 8.3 ml 3M NaOAc (to
0.1 M final[1:30 dilution]). Mix.
7. Add 625 ml (2.5 volumes) of cold 95% EtOH. Mix by inverting
8. Put at -20ºC for 10 minutes
9. Spin in microfuge in cold room, 15 minutes
10. Decant, resuspend pellet in 100 ml loading buffer
RNA isolation buffer: for 50 ml
0.5 ml NaCl 5 ml 5M NaCl
0.2 M TRIS/HCl, pH 7.6 5 ml 2M TRIS/HCl
0.01 M EDTA 1 ml 0.5M EDTA
1% SDS* 5 ml 10% SDS
*
SDS will precipitate out- warm ~15 sec. in microwave
RNA loading buffer (same as sequencing STOP buffer)
9.5 ml deionized formamide
400 ml 0.5 M EDTA
pinch of Bromphenol Blue
pinch of xylene cyanol
32
P Sequencing1. Labeling primer
Oligo (0.5-1 pmol/ul) 2 m l
10X kinase buffer 0.5 m l
g -32P ATP 0.4 m l
T4 kinase (1U/m l) 0.1 m l
Sterilized H2O 2 m l
Total volume 5 m l
*Add the T4 kinase next to last and add 32P last
Incubate at 37° C for 30 minutes
Heat to terminate reaction at 68° C for 3 minutes
Put on ice
2. Pre-reaction mixture
Per DNA
Labeled primer 5 m l
10X Taq sequencing buffer 4.5 m l
Sterilized H2O 23 m l
Taq DNA polymerase (5U/m l) 1 m l
Total volume 33 m l
Make an n+1 stock solution of pre-reaction mixture*Multiply each volume by n+1 (where n is the number of STET DNA’s) and make a stock solution.
3. Place 1-3 m l of each STET DNA (~50 fmol DNA) to be sequenced in separate tubes
4. Add 33 m l of pre-reaction mixture into each tube of STET DNA and pipet to mix. Keep on ice.
5. For each STET DNA, set up 4 thin-walled PCR tubes – label A, C, T and G
Into each of the A tubes, place 2 m l termination mix A. Into each of the C tubes place 2 m l of termination mix C. Follow same procedure for T and G.
6. Add 8 m l pre-reaction mix to each tube set up in step E. Mix well.
7. Place one drop of oil in each tube and put in PCR machine.
25 cycles with 1 min 95° C
2 min 55° C
3 min 70° C
8. When cycles completed, add 5 m l stop buffer to each reaction and store at -20° C.
33
P SequencingNucleotide master mix
G |
A |
T |
C |
|
| Nucleotide master mix dGTP |
|
|
|
|
| 33P ddNTP | 0.5m l x n |
0.5m l x n |
0.5m l x n |
0.5m l x n |
n = number of STET DNA
Aliquot 2.5m l of master mix into n number of tubes.
Reaction mixture
For multiple (n) reactions with different primers or different STET DNAs prepare an n+1 stock of reaction buffer, H2O, and polymerase and aliquot. Then add unique primer and/or template in the appropriate concentration and volume to aliquots.
Reaction buffer 2m l
STET DNA _m l (50-500ng or 25-250fmol)
Primer _m l (0.5-2.5pmol)
Sterilized H2O _m l (to adjust total volume to 20m l)
Thermo Sequenase polymerase 2m l (add LAST)
(4U/m l)
Total volume 20m l
Cycling termination reactions
For each STET DNA, transfer 4.5m l of reaction mixture to each termination mixture (G,A,T,C) prepared in step 1. Mix well.
Place one drop of oil in each tube and place in PCR machine.
Run 25 cycles with 30 sec at 95°
30 sec at 55°
2 min at 72°
(Note: longer extension time may be required when sequencing with dITP)
4. When finished running, add 5 m l stop buffer to each reaction and store at -20° C.
35
S SEQUENCING1. Place 7 ml of STET DNA (from a prep resuspended in 25-30 ml) in a microfuge tube. Add 1 ml of 10 mg/ml Rnase A and incubate for 15 minutes at room temperature.
2. Add 1 ml primer (10-50 ng) and 1 ml of 1 N NaOH (fresh 1:10 dilution of 10 N stock).
3. Add 4 ml of TDMN* (with DTT). Incubate 10 minutes at room temperature.
4. Remove 10 ml and place in a fresh microfuge tube. Incubate on ice until ready for use.
5A. Make up labeling cocktail according to the number of reactions being run:
: |
1 rxn |
5 rxns |
10 rxns |
15 rxns |
| 0.1 M DTT | 1 ml |
5 ml |
10 ml |
15 ml |
| †diluted labeling mix | 2 ml |
10 ml |
20 ml |
30 ml |
| 35S-dATP | 0.5 ml |
2.5 ml |
5 ml |
7.5 ml |
| §diluted sequenase | 2 ml |
10 ml |
20 ml |
30 ml |
†dilute labeling mix 1:5 in H2O
§dilute sequenase 1:8 by mixing 1 ml sequenase (blue-cap), 0.5 ml pyrophosphatase (purple cap), and 6.5 ml enzyme dilution buffer. Sequenase and pyrophosphatase are in the enzyme box in the -20C freezer.
5B. Put 2.5 ml of each termination mix into four separate tubes:
G: green; A: yellow; T: purple; C: blue
(one set of four tubes is needed for each DNA that is being sequenced).
NOTE: Steps 5A and 5B can be done while the DNA preparation steps are incubating.
6. Add 5.5 ml labeling cocktail to each DNA tube. Incubate 3 minutes at room temperature. (If multiple reactions are run, space at 20 second intervals)
7. Transfer 3.5 ml of DNA/labeling cocktail mix to each of the termination mixes. Incubate 5 minutes at 37ºC. (If multiple reactions are run, maintain 20 second intervals between each set of four termination tubes)
8. Add 4 ml of STOP buffer to each tube. Put on ice or freeze.
9. To denature DNA, heat reactions for 2 minutes at 95ºC, then put on ice before loading on the gel. Load 2-4 ml of each reaction.
Solutions:
*TDMN stock
(store at -20ºC in 95 ml aliquots)
for 10 mls:
0.64 g TES (free acid, Sigma T-1375)
0.1 ml concentrated HCl
0.8 ml 1M MgCl2
0.4 ml 5M NaCl
to 10 ml with milli-Q H2O
before use, add 5 ml of 1M DTT to 95 ml TDMN stock
1M DTT (FW 154.2):
dissolve 0.154 g in 1 ml H20
store as 5 ml aliquots in box with TDMN
STOP buffer:
9.5 ml deionized formimide
400 ml 0.5M EDTA (20mM final)
50 ml of 10% bromphenol blue/10% xylene cylanol (0.5% final)
STET prep
(rapid plasmid mini prep)
Grow a patch of E. coli strain on LB/carb plates (6 sectors per plate)
Label microcentrifuge tube with the corresponding plate sector number)
Using a sealed capillary pipet, transfer the cells to 500 ml of STET buffer
Vortex to resuspend on multi-vortexer (no longer than 5 minutes)
Add 25 ml of lysozyme (10 mg/ml in STET buffer)
Boil immediately in the 100ºC heat block for 3 minutes (no longer!)
Microfuge for 15 minutes at room temperature
Remove pellet (goober) with a toothpick and discard. Add an equal volume (500 ml) isopropanol to the supernatant. Mix by inverting.
Microfuge for 10 minutes. (Be sure hinge of microfuge tube is pointing outwards so you know where to look for your pellet.)
Decant isoproponal. Carefully rinse pellet with 70% ethanol. Decant.
Dry pellet and resuspend in 50 ul of TE.
STET buffer DO NOT AUTOCLAVE!
for 500 ml for 100 mls
8 % sucrose 40 g 8 g (8% final)
5% Triton X-100 25 g 5 g
50 mM EDTA 9.3 g 1.86 g
50 mM Trizma base 3.03g 0.605 g
Note: May appear slightly cloudy: Its OK.
TCA precipitation of VLP fractions
(to concentrate proteins)
Place 100 ml of a fraction (or 2x50 ml if pooling fractions) in a microcentrifue tube.
Add 800
ml ice-cold H2OAdd 100
ml 100%TCA (located in hood)Mix. Incubate on ice 20’ (can also be left overnight)
Pellet in microfuge, full speed, 15’
Aspirate supernatent
Resuspend in 15
ml 1x protein sample buffer (containing bromphenol blue) Note: If sample turns orange/yellow after adding sample buffer, its because its very acidic. Add 2M TRIS (not pH’d) 0.5 ml at a time until sample turns blue again.Remember to boil sample 3 minutes before loading on gel.
2X protein sample buffer:2.0 ml glycerol
1.0 ml 2-ME
0.625 ml 2M TRIS, pH 6.8
1.375 ml H2O
5.0 ml 10% SDS
Yeast cell growth and induction for VLP preps
The basic procedure is to grow the cells in raffinose to use up internal glucose stores, followed by galactose induction. This is done as follows:
Preparation: Grow cells on glucose-drop-out plates to maintain the desired plasmid(s). Start by streaking the cells down the center of the plate and grow for 1-2 days at 30° C. Then spread the streak over the entire plate and grow for 2 days at 30° C.
The selective media to be used can be made according to the recipes in Table I. A 2X stock aliquoted and autoclaved in 500 ml bottles is convenient method and extra media is easy to store (see Table 1 footnote). YNB/CAA is the best media to use. This can be used if the only selection needed is -ura, -ade, and /or -trp (Don’t forget to add any of these that are not covered by the strain!). If any other selection is needed, drop-out media must be used.
Cell inoculation and growth: Prepare 450 ml of the appropriate raffinose media in a 2 L flask. Scrape the cells from the drop-out plates into the liquid media. The inoculated culture should be at an OD600 of » 0.5 (+/- 0.1). Record the OD reading and subsequent readings on the DATA SHEET. Grow the cells in the air shaker at 30° C for 6 hours. Record the OD600 and be sure that the cells are growing. There should be a slight increase in the OD600 in the time of inoculation. If this is not apparent, check the cells in the scope and make sure that most have small buds. (If not, something is wrong with the media.) Then add 50 mls of 20% galactose, and grow the cells at 22° C for the desired amount of induction time (standard time is 24 hours). Record the OD600 (a dilution of at least 1:10 will be necessary to get a meaningful OD reading). The cells should have reached an OD600 density comparable to values listed in Table 2 for that given strain.
Cell harvest: Pour the cell culture into a 500 ml Beckman centrifuge bottle - be sure that the inner cap has a black o-ring. Spin for 10’ at 5,000 rpm in the big GS-3 rotor. Wash the cell pellet once in 50 ml of H2O (in a 50 ml blue-cap falcon tube). Spin 10’, 4,000 rpm in a table top centrifuge. Decant well. Resuspend the pellet in 5 ml cold Buffer B. Measure the total volume. The desired volume is 7.0-8.0 ml total (5 ml buffer + 2-3 ml cells). If the volume is greater, add another 5 ml buffer B. Freeze 7-8 ml aliquots of the cell suspension in 50 ml Falcon tubes (blue cap, the orange ones tend to break), recording the strain name, the date, and the total mls of packed cells on the tube. Also record this information on the VLP DATA SHEET. Frozen cells have been stored at -80° C for a year without loss of VLP activity.
YEAST TRANSFORMATION
Sterile Technique!
1. Inoculate a colony of yeast into 5-10 ml rich medium.
2. Grow overnight, rotating at 30°C.
3. Pellet cells in table top centrifuge 4 minutes, 1500 rpm and wash 1x in 5 ml sterile H2O.
4. Pellet cells (as in 3) and wash in 5 ml 0.1 M LiAc/1xTE.
5. Pellet cells (as in 3) and resuspend in 1/10- 1/100 volume (200-500
ml)LiAc/TE. (Be sure to wipe off the outside of your pipetman with 70% EtOH)6. Aliquot cell suspension, 100
ml per sterile microfuge tube.7. Add 20
mg (2 ml of 10 mg/ml) boiled carrier DNA to each tube. Use one tube as a NO DNA control. Add transformation DNA (up to 10 ml of STET) to the remaining tubes as needed.8. Incubate 5-10 minutes, room temperature.
9. Add 500
ml of PEG/LiAc/TE, mix, incubate 30-45 minutes at 30°C.(optional, in addition to PEG, add 50
ml of 1 M DTT (100 mM final) for increased efficiency)10. Heat shock 5-15 minutes at 42°C. (15 minutes for strains of low transformation efficiency)
11. Spin, resuspend in 500
ml sterile dH2O and plate on selective medium:ml on one plate-NO DNA control: plate all 500
-DNA sample: plate 50
ml and 450 ml on two separate plates.
50 ml 0.1 M LiAc/1X TE 10 ml PEG/0.1 M LiAc/1x TE
5 ml 1 M LiAc 1 ml 1 M LiAc
0.5 ml 100X TE 0.1 ml 100X TE
45 ml H2O 9 ml 44% PEG
VLP prep
Before beginning:
Make up sucrose step gradient 20%/30%/70% of volumes 15 ml/5 ml/5 ml respectively. Place gradients in cold room.
1. Thaw a tube of 1M DTT stock and a tube of 100 mM PMSF
2. Defrost cells in beaker of lukewarm water. Keep cells cold during thawing by placing intermittently on ice. When cells are thawed, place tube on ice.
3. Add 25
ml 1M DTT to cells and swirl to mixAdd 80
ml 100 mM PMSF to cells and swirl to mixGO TO COLD ROOM
4. Add cold glass beads to tube until level with liquid. Place on multivortexer for one minute. Top off with beads as needed. Vortex for 15 minutes
5. While cells are vortexing:
ml DTTA. prepare wash buffer: (multiply times number of gradients)
10 ml Buffer B/EDTA
30
100
ml PMSFB. Label corex tubes and place on ice in cold room
C. Put SS34 rotor (with inserts) in superspeed to cool down
6. Check cells for breakage by phase-contrast. Note ~% breakage in notebook.
7. Collect supernatant with glass Pasteur pipette into 15 ml corex tube.
8. Add 3.0 ml Buffer B/EDTA/DTT/PMSF to cells & beads. Vortex and shake well. Pool supernatant in corex tube.
9. Repeat wash with 3.0 mls additional of Buffer B/EDTA/DTT/PMSF.
10.. Check that samples are of same volume (visual OK). Adjust as needed with Buffer B/EDTA/DTT/PMSF. Cap with parafilm and mix by inverting.
11. Spin 10K, 10 minutes, 4°C in SS-34 rotor
12. Carefully pull off supernatant (no "mud") and place into a 15 ml polypropylene tube. Adjust to 8 mls with Buffer B/EDTA/DTT/PMSF.
13. Transfer ~7.5 mls of liquid to the sucrose gradient, so that sample is within 2-3 mm of tube rim.
14. Balance tubes to +/- 0.02 g.
15. Spin gradient in swinging bucket rotor, 25K, 150 minutes, 4°C.
While gradient is spinning label tubes for fraction collection and place on ice in cold room.
Fraction collection (in cold room):
Clamp tube onto ring stand and pierce bottom of tube with an 18-gauge needle using a twisting motion, until sample begins to drip out.
Collect 1.2 ml fractions with tubes on ice. Ideal rate is ~14 seconds per fraction. Leave fractions on ice after collection.