1) Add cored single plaques (1 plaque = 107 phage) to 0.5ml fresh cells (ON culture in LB w/ 10mM MgSO4 0.2% maltose) in 50ml copolymer tubes. Make also mock - plaque.
2) inc. 15\' 37C
3) add 5ml LB + 5mM CaCl2
4) Shake hard 37C 4-5hrs until cleared w/ cell debris -compare to control
5) add 100ul ChCl3, shake 5\' 37C
6) spin 3000rpm table top 10\'
7) transfer sup to corex, add 4ml 20% PEG,2M NaCl, invert well, inc 60\' on ice.
8) spin 3000rpm tabletop 20min, drain and wipe tube dry.
9) resuspend pellet in 750ul LB, transfer to eppi, add 750ul LB/DE52, invert 20- 30x.
10) spin 5\', transfer sup, spin again, transfer sup.
11) add 7ml sup 13ul 0.1mg/ml Poteinase K, 32ul 10% SDS, mix inc RT 5\'
12) add 130ul 3M KOAc, inc 88C 20\', cool on ice 10\', spin 10\' 4C
13) transfer 800ul and add 800ul -20C isopropanol, inc -70C 15\', spin 10\'
14) decant well and briefly speedvac pellet, resuspend in 20ul TE.
LB/DE52
Wash (mix/settle/aspirate) 50g Whatman DE52-cellulose in 100ml blue cap with 0.05
N HCL till pH under 4.5. Add 10n NaOH dropwise w/ stirring till pH 7. Wash 3x w/ LB. Add LB to 25% final vol w/ 0.2% NaN3. store 4C LEAF PCR PROTOCOL
(Klimyuk et al., 1993, TPJ 3: 493-494)
1) Samples are harvested in 1.5 ml tubes and stored on ice.
2) 40ul of 0.25N NaOH was added and the samples boiled for 30 sec.
3) 40ul of 0.25N HCl then 20ul Tris mix was added and the samples boiled for another 2 min.
-tissue samples can then be used immediatly or stored at 4 C for several weeks.
-The amount of tissue used in each PCR reaction should not exceed 2mm2 or the reaction will not work. A small amount of treated material can be excised for use in a PCR reaction with a sterile Gilson tip.
PCR reaction conditions are as follows:
total volume= 50ul
for 5.5 reactions
10X buffer 5ul 27.5uls
10mM dNTPs 1.25uls 6.875uls
primer A 2.5uls 13.75uls
primer B 2.5uls 13.75uls
dH2O 38.75uls 213.1uls
taq poly 1.0ul 5.5uls
95 C 10min 1X
95 C 30sec
55 C 30sec
72 C 45sec 30X
72 C 10min 1X
run 15ul on a 2% agarose gel
note: 2.5 times more primer is used and 2 times more taq polymerase in the leaf PCR protocol. If you could get by with less, Jonathan Jones would have done so!
Stocks
0.25N HCl
0.25N NaOH
Tris buffer:
0.5M Tris pH 8.0
0.25% Nonidet P-40
LEAF PCR ON ARABIDOPSIS TISSUE WITHOUT ALKALINE TREATMENT
Preparation of Master mix:
1 x Taq-buffer
1.5 mM MgCl2
200 mM of each dNTP
1 mM each primer
0.5 ml 20 x Taq polymerase
The mix is stored on ice until use.
Preparation of leaf tissue:
Put the leaf in a small Petri-dish. Make a hole in a leaf with the narrow end of the Pasteur pipette (a forceps might be helpful) and place the leaf in a PCR-tube, if necessary by blowing. On ice, add 50 ml the Master mix.
Running the cycles:
Transfer the tubes directly from ice to the prewarmed 94 C block on the Robocycler and run the following cycles:
94 C for 3 min 1 x
94 C for 30 s; Tann.* for 1 min; 72 C for 1 min-1 min 30 s 35 x
72 C for 10 min (optional) 1 x
Appropriate controls:
Positive Negative
For screening transgenics: plasmid ColO
ColO with endogenous primer-set gDNA
gDNA with endogenous primer-set - DNA
*The annealing temperature (Tann.) should be 2-3 C below the calculated Tann.. MAXAM GILBERT SEQUENCING
NEN kit #NEK010
1)Modification reactions: volumes are in ul, DNAs are 10 20k cpm/ul
Do G, then T C and C while A G is incubating
G react A G react T C react C react200 G buf 10 H2O 10 H2O 15 5M NaCl5 DNA 10 DNA 10 DNA 5 DNA1 DMS 2Pip For 30 hydrazine 30 hydrazine2\', RT 15\', 37C 5\', RT 5\', RT50 DMS stop snapfreeze 200 hz stop 200 hz stop750 EtOH 750 EtOH 750 EtOH5\' spin 5\' spin 5\' spin
2)Cleavage reaction: speedvac all samples
resuspend in 100ul 1M piperidine (diluted from 10M stock)
inc 30\', 90C, tighly sealed
3)Cleanup
+ 1ml butanol, vortex
spin 3\', aspirate
+ 150ul 0.1% SDS, vortex
+ 1ml butanol, vortex
spin 3\', aspirate
+ 500ul EtOH
spin 3\', aspirate
speedvac
4)PAGE, see ureapage.ptc MICROSOMAL PREP
from Halkier M ller 1989 Plant Physiol. 90, 1552-9
1) 0.1-1g sample freeze-dried material powdered in motar/pestle under lN, suspended in 20xv/w MEB (microsome extract buffer):
250mM sucrose
100mM Tricine (7.9)
50mM NaCl
2mM EDTA
2mM DTT
100mg/20ml PVPP (BDH Polyclar AT or Sigma 6755)
0.8mM PMSF
5ug/ml antipain leupeptin
2) Spin 10\' 10,000g
3) Transfer supernatant w/ pippette to black double capped polycarbonate tubes
(16x76mm, Cat # 355603). Save pellet.
4) Spin supernatant 60\' at 165,000g (That\'s 70krpm in a 70ti).
5) Pippette off supernatant and save.
6) Resuspend microsomal pellet in 0.1-1ml MEB. Dyalyze against
50mM Tricine (7.9)
2mM DTT
0.8mM PMSF MINIPREP - ALKALINE LYSIS
1) 1.5ml o/n culture to eppi, spin 1\', aspirate sup
2) resuspend by vortex 5\' RT in 100ul miniprep solution 1 MPS1
3) + 200ul MPS2, invert tubes rapidly 3x, inc 5\' ice
4) + 150ul MPS3, vortex upsidedown 10\", inc 5\' ice
5) spin 5\' RT
6) transfer to eppi - 7a) for sequencing
7) PCHCl3 ext
8) spin 2\' RT
9) transfer eppi
10)+ 900ul EtOH
11)inc 2\' RT
12)spin 5\' RT
13) aspirate
14) 70% EtOH wash spin
15) aspirate, speedvac
16) resuspend in 50ul TE, use 2 for digests
7a) +900ul EtOH
8a) spin 5\' RT
9a) aspirate
10a) + 1mL 70% EtOH, spin
11a) aspirate, resuspend in 200ml TE, 2mg RNAseA, inc 15\' 37 C
12a) Phenol/CHCl3 extract, add 20ml 3M NaOAc, EtOH ppt, 70% wash
13a) resuspend in 30ml TE
14a) see sequenase protocol for denaturation
16) resuspend in 50ul TE
solutions:
MPS1, frozen stock /50ml
50mM glucose 2M 1.25ml
10mM EDTA 0.25M 2ml
25mN Tris 8 1M 1.25ml
MPS2, fresh /10ml
0.2N NaOH 10N 200ul
H20 - 8.8ml
1% SDS 10% 1ml
MPS3 /100ml
60mM KOAc 5M 60ml
1.2M HAc [] 11.5ml
H2O - 28.5ml NEPHGE NONEQUILIBRIUM PH GEL ELECTROPHORESIS
1) Gel tubes are 0.25 x 13.5cm, acid (1/1 H2SO4/HNO3) washed,rinsed, silated, rinsed EtOH, H2O, baked, one end plugged w/ 2xparafilm
2) make gels afternoon of day before electrophoresis
3) make up gel solution
4) polymerized w/ 20ul fresh 10% APS, 7ul TEMED
draw into syringe fitted with 23 gauge and PE tubing, add frombottom to top.
Aspirate solution back down to 1.5cm from top toclear meniscus
5)overlay w/ H2O 1 2h while polymerizing, replace w/ 20ul samplebuffer o/n, parafilm top
6)secure dialysis tubing soaked in lower buffer to bottom of gelsw/ tubing slice.
Avoid bubbles, best to do this w/ end submergedin lower reservoir
7) aspirate sample buffer from gel top
8) load sample (10ul), cover w/ 10ul overlay buffer, upper bufferto top
9) connect electrode to bottom, electrophorese 400v for 2400Vh
10) aspirate upper buffer, then overlay, then sample, rinse topof gel w/ sample buffer, then fill to top w/ SDS sample buffer
11) expel gel from tube slowly using tubing fitted syringe intofalcon tubes w/ 5ml SDS sample buffer.
12) equilibrate w/ rocking 1.5h
13) Load on 7.5-15% SDS-PAG (notched plates) w/ sample buffercontaining 1% pre electrophoresed agarose. MW standard wells arecast on left side with plastic teeth. Include BPB in some of theagarose. Electrophorese at 20mA, 16h
Sample prep: salt is not allowed!
lysate wgerm, 5ul lysate proteins, 20 100kcpm protein, 50 200ug
solutions:
Gel, fresh
[final] [stock] /10ml
urea 9.2M 5.53g
NP 40 2% 10% 2ml
Acryl/Bis 4% 28.4/1.6% 1.33ml
Amphol 3.5-9.5 1.86% 40% 465ul
Amphol 5.0-7.0 0.14% 40% 36ul
methylamine 10mM 7.8ul
Ampholines are LKB 1818 101 1809 121
Sample buffer, frozen 1ml aliquots
urea \" \" \"
NP40 \" \" \"
amphol 3.5 9.5 \" \" \"
amphol 5.0 7.0 \" \" \"
methylamine \" \" \"
DTT 10mM 1M 0.1ml
Overlay buffer, frozen 1ml aliquots
urea 8M 4.81g
amphol 3.5-9.5 0.2% 40% 50ul
methylamine 10mM 7.8ul
SDS sample buffer
glycerol 10% 10ml
Bme 5% 5ml
SDS 2.3% 10% 46ml
Tris 6.8 2.5M 0.5M 12.5ml
Upper buffer
36.6M, 2ml/l
Lower buffer
83.5mM ethanolamine, 5ml/l NORTHERN BLOT IN FORMALDEHYDE GEL
gel prep:
1) add 2g agarose to 143ml H20, microwave, put at 60C
2) + 20ml 10x northern running buffer NRB at 60C
3) + 37ml formaldehyde at 60C
4) cast well mixed in fume hood
sample prep:
1) mix RNA + H20 4.5ul
10x NRB 2ul
formaldehyde 3.5ul
formamide 10ul
2) heat 60C 15\'
3) quickly + 2ul/northern sample loading buffer NSLB
4) quickly load
5) run 2 h 100-150v in 1x running buffer NRB
6) when run complete, soak gel 1h in 20x SSC
7) transfer to Nc in 10x SSC
8) prehyb 6h, hyb 40h, 42C in PB HB (see colony hybridization)
9) wash filters 2x 55C, 1xSSC,1% SDS, 20\'
Nx 55C, 0.1xSSC, 0.1% SDS, until low background solutions NRB, autoclave, turns yellow
0.2M Mops 7
50mM NaOAc
5mM EDTA
NSLB, DEPC H2O, filter sterilize
50% glycerol
1mM EDTA
0.4% BPB
0.4% XC
MW markers:BRL RNA ladder Cat # 5620SA NUCLEAR EXTRACT PREPARATION
Based on Parker Topol (1984) Cell 36 357 369, Green et al.(1987) EMBO J. 6 2543 2549) using swelling in low osmoticum andlysis at high osmoticum. AmSO4 ppt concentrates and separateshistones from binding proteins. All steps in cold room on ice.Volumes here for 100mg nuclear protein (see nucprep.ptc).
1) thaw frozen nuclei on ice
2) add 1 vol NWB triton, transfer to 15ml corex
3) spin 5\' 3krpm
4) decant supernatant, drain well
5) resuspend in 7ml total of nuclear lysis buffer NLB, transferto Beckman ti70
polycarbonate tubes (16x76mm, Cat # 355603)
6) add 770ul 4M AmSO4
7) rock 20\', should see viscosity increase
8) spin 1h, 35krpm ti70. The pellet is hardish, supernatantviscous w/ DNA
9) add 0.3g/ml AmSO4 to supernatant w/ stirring in a 25ml beaker.It takes at least
0.5h to dissolve
10)spin 15\', 10krpm in 15ml corex. The supernatant containshistones, the pellet primarily binding proteins. Decant thorough ly, proteins in supernatant can be ppted w/ 80% AmSO4
11)add 250ul nuclear extract buffer NEB to pellet,stand on ice5\', gently resuspend and transfer to dialysis tubing (cut off14kD, no azide)
12)dialyze 4h x 2 x 2l NEB
13)spin 10\', 12krpm, l N2 freeze supernatant and store 70C
14)yeild for pea is 5-10% of nuclei prep protein, 2-5% for rice wheat. Good extracts are 5-10mg/ml
Solutions: autoclaved, precool, add DTT, Bme and Pase inhibitorsprior to use
4M AmSO4, BRL enzyme grade
NLB Stocks/100ml
110mM KCl 3.7ml 3M
15mM Hepes/KOH 7.6 7.5ml 0.2M
5mM MgCl2 500ul 1M
1mM DTT 100ul 1M
5ug/ml antipain leupeptin 100ul 5mg/ml
NEB
40mM KCl 1.2ml 3M
25mM Hepes/KOH 7.6 12.5ml 0.2M
0.1mM EDTA 50ul 0.2M
10% glycerol 10ml
1mM DTT 100ul 1M for resuspension
5ug/ml antipain leupeptin 100ul 5mg/ml for resuspension
5mM Bme 30ul for dialysis
0.8mM PMSF 1ml 80mM for dialysis NUCLEI PREP
Based on Watson Thompson (1986) Methods Enzymol. 118 57 75,Green et al. (1987) EMBO J 6 2543 2549, and Kannangara et al.(1977) Carlsberg Res. Commun. 42 431 434.
Set up cold room daybefore, all solutions at 4C, all tubes on ice.
1) 2.5l (15-20 trays) of seed is grown 7 d in the dark (givesless starch) in vermiculite w/ occassional H2O. Harvest w/ scis sors to yeild approx. 1kg leaves.
After weighing, leaves arefurther cut to approx. 2 cm length.
2) wash half in 4l H2O
3) rinse w/ 1.5l homogenization buffer HB
4) transfer to large razor blender, add 2l HB
5) smash w/ 3 1\" bursts, then 15\" low speed
6) filter thru 1000u and 80u nytex screens (see diagram)
7) rinse blender w/ 500ml HB, brush lower screen to free debris
8) spin 15\' (time to smash other half of material) at 3k rpm in 8250ml swing out fuge tubes
9) decant resuspend gently (only wide mouth pipettes) in 5ml/t ube (80ml total) nuclei wash buffer NWB + Triton, transfer to 430ml Corex tube and wait for next smash
10) spin corex tubes 5\', 3k rpm in swing out
11) resuspend gently with small brush in 10ml NWB Triton/tube(40ml total)
12) spin 5\', 3k rpm
13) for storage, resuspend in 4ml NWB Triton, mix w/ 1.6mlglycerol,freeze in lN2 and store at -70C
14) take 1 5ul for Bradford assay. Yeild for rice approx 80-120mg, twice that for pea
Solutions: autoclaved, precool, Bme and Pase inhibitors addedprior to use Homogenization buffer HB stocks/l
1M hexylene glycol(Aldrich) 127.5ml 7.8M
10mM Pipes/KOH 7 10ml 1M
10mM MgCl2 10ml 1M
0.5% v/v triton X 100 25ml 20%
5mM Bme, 300ul
0.8mM PMSF 10ml 80mM
Nuclei wash buffer NWB
same as HB but
0.5M hexylene glycol
w/ or w/out triton NUCLEAR PREP FROM PROTOPLASTS
1) 8x106 protos in cellwells, collect and spin in 2x 15ml APM at 500rpm for 5\' in bench fuge to pellet. decent supernatant. Subsequent steps at 4 C. Examine cells here, and after next step to check lysis.
2) Lyse by resuspending in 50ml ice cold NIB.
3) Filter through a 20m screen, rinse sreen w/ 50ml NIB, pool in 3x 30ml corex tubes, 1 tube balance.
4) Pellet nuclei in HB4 swingbucket at 2470rpm (1000g), 20\', 4 C.
5) Gently resuspend in 40ml NIB, pool in 2x 30ml corex.
6) Spin to pellet starch and debris in HB4 at 110rpm (1g), 3\', 4 C.
7) Transfer supernatant containing nuclei to new tubes. Pellet at 1750rpm (500g)
15\'. Decant completely, quickly wipe tube dry.
8) Resuspend nuclei in 5ml HEN-5. Transfer to ultra tube.
9) Add 0.55ml 5M NaCl ([0.5M] final) and 27.5ml 1M spermidine ([5mM] final).
10) Gently agitate at 4 C, 2hr.
11) Spin clear in Ti50 rotor at 37.5k rpm (88kg) for 15\', 4 C.
12) Transfer supernatant to fresh ultra tube. Add 0.45g/ml finely crushed AmSO4, gently agitate at 4 C for 30\' after AmSO4 fully dissolved.
13) Pellet in Ti50 at 28k rpm (50kg) for 30\', 4 C.
14) Resuspend in minimal volume HEN-20. Try 50ml, followed by 50ml to rinse tube.
15) Dialyze in small tubes against 2 x 100ml HEN-25 for 2x 3hr.
17) lN2 freeze in 20ml fractions, store at -70 C.
NIB stock ml/l
10mM TRIS-HCl 7.5 1M 10
10mM KCl 2M 5
10mM MgCl 1M 10
10mM me 14.27M 700ml
20% Glycerol 100% 200
0.02% Triton X-100 100% 200ml
HEN stock ml/l
20mM HEPES-KOH 7.5 0.5M 40
1mM EDTA 250mM 4
50mM NaCl 5M 10
5-20% Glycerol 100% 50-200
1mM DTT 1M 1 OLIGO DT CELLULOSE PURIFICATION OF MRNA
Cat # 20003, oligo(dT) cellulose type 3, Collaborative research Inc., 128 Spring
St. Lexington Mass 02173
Column prep: suspend cellulose in elution buffer, wash in dis posable spin columns set in falcon tubes w/ 10 volumes binding buffer
Binding buffer: 10mM Tris 7.5
500mM NaCl
1mM EDTA
0.5% SDS
Wash buffer: same SDS
Elution buffer: 10mM Tris 7.5
1mM EDTA
