Protocol English PMB Lab Prof. Yang

Protocol of PMB Lab - Chonbuk National University
Preparation of Competent E.coli - Ⅱ

(CaCl2 method)
1. Inoculate 5㎖ LB medium with single colony and overnight culture

at 37℃, 200rpm.
(If the cell carry some resistant gene add antibiotics)
2. Next day, inoculate 1㎖ overnight cultured cell to 100㎖ fresh
medium(LB) in a 500㎖ flask.
LB media
bacto-tryptone 1g
yeast-extract 0.5g
NaCl 1g
/100㎖
3. Grow up it until OD@600 = 0.5 (200rpm, 1.5∼3 hrs) at 37℃.

4. Pour it to two 50㎖ tubes and spin 4000rpm for 5min at 4℃.
5. Decant the medium from the cell pellets. Stand the tubes in an inverted
position on a pad of paper towels for 1 minute to allow the last traces of
media to drain away.
6. Suspend each pellet with 12.5㎖ 0.1M CaCl2 and leave it in ice for
15min.
0.1M CaCl2 0.735g/50ml and Filter sterile
7. Spin 4000rpm for 5 min at 4℃.

8. Decant the medium from the cell pellets. Stand the tubes in an inverted
position on a pad of paper towels for 1 minute to allow the last traces of
media to drain away.
9. Suspend each pellet with 2ml 0.1M CaCl2 and 300㎕ autoclaved
Glycerol and leave it in ice for 2hr.
10. Distribute 200㎕ cell to the pre-chilled EP tubes(do it in cold room),
freeze them in liquid Nitrogen(LN2) and transfer to storage container in -
70℃.
Lab of Plant Molecular Biology - Prof. Yang Moon Sik (Chonbuk National University, Korea)
Transformation
1. Throw competent cell in ice.
2. Aliquot 10㎕ of ligation mixture to a new tube.
3. Add 200㎕ of competent cell into a ligation mixture.
4. Inoculation in ice for 30min ∼ 1hr with intermittent tapping.
5. Heat shock at 42℃ for 90sec.
6. Put the tube into ice for 3mins.
7. Add 800㎕ LB liquid medium and then incubation at 37℃ for 1hr.
8. Spreading onto plate (containing your favorite antibiotics).
9. Leave the plate at room temperature until the liquid has been absorbent.
10. Invert the plate and incubate at 37℃ for 12 ∼ 15hrs.
----------------------
One step miniprep method

for the isolation of plasmid DNA
1. Inoculation of transformant in a EP tube (LB + antibiotics, 0.5㎖).
2. Add 0.5㎖ of phenol:chloroform:isoamylalcohol(25:24:1).
3. Mix by vortex at the maximum speed for 1min.
4. Spin at 12000rpm for 5min.
5. After the spin, remove carefully about 0.4㎖ of the supernatant into
new EP tube.
6. Add 0.4㎖ of the isopropanol and mix well.
7. Immediately, spin at 12000rpm for 5min.
8. Pour off the supernatant and wash the pellet with 70% EtOH(0.5㎖).
9. Vacuum dry the pellet.
10. Suspend in 20㎕ of TER(TE+20㎍ RNAse/㎖).
11. About 5㎕ of this DNA can be cleaved with appropriate restriction
enzymes for analysis.
----------------------
Mini-Prep of plasmid DNA (Alkaline lysis method)
1. Inoculate 5 ml culture of the proper medium (ie. with antibiotics) from a

single colony.
Grow at 37℃, overnight at 200rpm. Mark the EP tubes on top and on side!!!
2. Transfer 1.5ml EP tube, spin 15sec.. Decant the supernatant.

Repeat this until all of the 5 ml culture is centrifuged. Wipe the inside wall of
the tube with a KimsWipe.
3. Suspend the pellet in 0.1 ml Sol I (200 ul), vortex well and then standing at
RT for 10min
※ Sol I composition
50mM glucose
10mM EDTA
25mM Tris-HCl
4㎍/㎖ Lysozyme (Option)
4. Add 0.2 ml fresh Sol II (400ul) , mix well (not vortex) and put on the ice for
5min.
※ Fresh Sol II composition
0.2N NaOH 2N NaOH 100㎕
1% SDS 10% SDS 100㎕
DW 800㎕ / 1㎖
5. Add 0.15 ml of Sol III (300ul), mix well (not vortex) and put on the ice for
10min.
※ Sol III composition
5M KAc 60㎖
glacial HAc 11.5㎖
DW 28.5㎖ / 100㎖
6. Centrifuge at 12000rpm for 10min.
7. Transfer the supernatant into the new EP tube,

Add equal volume of Phenol(Phenol:CHCl3:Isoamylalcohol) soln.,
Vortex well for 30sec., centrifuge at 12000rpm for 5min.
8. Transfer the supernatant from centrifuged tube into the new tube.
Add equal volume of CHCl3. and vortex well for 30sec., centrifuge at
12000rpm for 5min.
8. Transfer the supernatant from centrifuged tube into the new tube
Add two volume of 100 % cold EtOH, mix well, incubate at -20℃ for 15min,
Centrifuge at 12000rpm at 4℃ for 15min , decant the supernatant.
9. Wash the pellet with 500㎕ of 70% EtOH (vortex to see the pellet swim.)
Suspend well and centrifuge at 12000rpm for 5min, decant the supernatant,
The pellet may be loose, be careful when you decant 70 % EtOH.
10. Vacuum drying the DNA pellet.
11. Dissolve the DNA pellet with TE buffer and store at -20℃.
Elution of DNA fragment using Geneclean kit (from Bio101)
1. Excise gel slice and place it in a EP tube
2. Add 3 volume of sodium iodide (100㎕=0.1g agarose)
3. Heat in a 45∼55℃ water bath for 5min to melt gel.

mix solution well for every 1min.
4. Add 5ul of glass milk, vortex 30sec and standing on ice for 10min
5. Centrifuge at 12000rpm for 20sec, discard supernatant.
6. Add 500㎕ of Washing buffer, suspend by pipetting back and forth)

Centrifuge at 12000rpm for 20sec, discard supernatant
→ 3 times ≪ last : throughly discard≫
7. Add any volume of TE or DW 20㎕, vortex softly
8. Heat in a 45∼55℃ water bath for 5min
9. Centrifuge at 12000rpm for 1min
10. Obtain the supernatant, transfer into new tube without glass milk.
----------------------
Triparental mating
1. Grow the cell in 5㎖ LB media with vigorous shaking at appropriate

temperature.
Add appropriate antibiotics and culture overnight.
A. tumerfaciens (100㎍/㎖ Rifampicine, 200㎍/㎖ Sm)

pRK2013 (helper plasmid, 50㎍/㎖ Kanamycine)
pBI121 (binary vector, 50㎍/㎖ Kanamycine)
2. Mix the three bacteria!!

① Each bacteria transfer 1.5ml EP tube, spin 30sec.. Decant the
supernatant.
② Add 1㎖ of LB,
③ ①, ② repeat twice, lastly remain the supernatant, suspend!
④ Add 100㎕ of each bacteria on the LB plate (no antibiotics)
Spreading until drying the surface of plate.
⑤ Incubation at 26∼28℃ for 1∼2days.
3. Select the bacteria using the antibiotics

① Add 1∼5㎖ of 10mM MgSO4, suspend the grown bacteria using
spreader.
② Aliquot 900㎕ of 10mM MgSO4 in the EP tube
③ Add 100㎕ of suspened bacteria in the first EP tube containing 900㎕
of 10mM MgSO4
Mix well and then second, third, forth..
Prepare original , 10-1, 10-2, 10-3, 10-4 serial dilution sample
④ Spread the 100㎕ of diluted sample on the LB plate

(100㎍/㎖ Rif, 50㎍/㎖ Km)
⑤ Incubation at 26∼28℃ for 2∼3days.
⑥ Inoculate a single colony in 5 ㎖ LB media of the proper medium
(ie. with antibiotics ) from antibiotics selected colony.
Grow at 26∼28℃ for 1∼2days.
----------------------
Agrobacterium plasmid quick screening
Modified Nucleic acid research(1979) 7:1513-1523
1. Grow Agrobacterium cell in 5㎖ YEP(or LB) with vigorous shaking at
28℃. (add appropriate antibiotics and culture overnight)

2. Transfer the culture to Ep tube and harvesting.
3. Suspend the cell pellet 100㎕ of cold SolⅠ(with 4㎎/㎖ lysozyme) and
incubate for at least 30min at room temperature.
4. Add 200㎕ fresh SolⅡ and shake to mix and incubate for 15min at room
temp.
5. Add 30㎕ phenol(equilibrated with 2 volume of SolⅡ) and vortex gently
for a few sec.
6. Add 150㎕ of 3M NaOAc(pH4.8) and shake the tube briefly.
7. Store the tube at -20℃ for 15min.

8. Centrifuge for 4min and transfer the supernatant to a new Ep tube.
9. Option : phenol and chloroform extraction.
10. Fill the tube with 2 volume of cold 95% EtOH. Store at -70℃ for
15min.
11. Centrifuge for 10min and discard the supernatant.
12. Add 500㎕ of 0.3M NaOAc(pH7.0) and 2 volume of 95% EtOH.
Store at -70℃ for 15min.

13. Centrifuge for 10min and discard the supernatant.
14. 70% EtOH washing and speed-vac dry.
15. Dissolve pellet in appropriate TE buffer.
Tobacco Transformation
- leaf disk transformation method -
1. Use mass to make leaf disks. size: 0.5㎠
2. Soaking leaf disks in the MS104 medium
3. Harvest the grown Agrobacteria, suspend the harvested cell with 1㎖ of MSO
medium
4. Inoculate Agrobacterium in the soaking leaf disks for 10min
5. Transfer to sterilized filter paper over MS104 plate.
6. Culture at 25℃ for 2∼3days in dark condition
7. Transfer leaf disks to MS selection medium (contain an appropriate

antibiotics)
8. Culture at 25℃ for 2∼4weeks in light condition. After 2∼4weeks, shoots

appear.
9. Transfer shoots to root-inducing medium. Roots appear.
10. Transfer to soil after 3 weeks. Get to regenerated plants
MSO Media
MSO medium MS104 medium MS selection medium MS rooting medium
MS salt 4.3g/ℓ MS salt 4.3g/ℓ MS salt 4.3g/ℓ MS salt 4.3g/ℓ
Sucrose 30g/ℓ Sucrose 30g/ℓ Sucrose 30g/ℓ Sucrose 30g/ℓ
Agar 0.8% Agar 0.8% Agar 0.8% Agar 0.8%
B5 Vitamin 1㎖/ℓ B5 Vitamin 1㎖/ℓ B5 Vitamin 1㎖/ℓ B5 Vitamin 1㎖/ℓ
BAP 1㎎/ℓ BAP 1㎎/ℓ
NAA 0.1㎎/ℓ NAA 0.1㎎/ℓ
Cefotaxime 500㎎/ℓ Cefotaxime 500㎎/ℓ

300㎎/ℓ
Kanamycine 100mg/ℓ Kanamycine 100㎎/ℓ
(nên làm)
Adjust to pH5.7 with 2.5M KOH
Stock solution 100㎎/㎖ myo-inositol

Vitamin B5 stock (1,000×)
10㎎/㎖ thiamine-HCl
1㎎/㎖ pyridoxine-HCl
1㎎/㎖ nicotinic acid

Adjust to pH5.7 with KOH.
Filter with 0.22㎛ milipore filter.
BAP(benzylaminopurine) Dissolve 10㎎ BAP in diluted NaOH.
stock (1㎎/㎖)
All the BAP was dissolved, add D.W to 10㎖.
Adjust to pH5.7.
NAA(naphthaleneacetic acid) Dissolve 10㎎ NAA in diluted NaOH or 70% EtOH.
stock (1㎎/㎖)
All the NAA was dissolved, add D.W to 10㎖.
Adjust to pH5.7.
Cefotaxime stock (250㎎/㎖) Add 4㎖ D.W to 1vial(1g) and dissolve.
Kanamycine stock (200㎎/㎖) Add 5㎖ D.W to 1vial(1g) and dissolve.
Stock Solution
Plant Rapid Genomic DNA Preparation

Salah M., NAR 25(22) : 4692∼4693 (1997); rapid.pdf
① Add Grind 300㎎ of fresh leaf to Ep tube with 400㎕ of homogenizing buffer
② Add 40㎕ of 20% SDS(final conc. is 2%), 8㎕ of proteinase K(20㎎/㎖, final conc.
is 400㎍/㎖) and 10㎕ of RNase A(10㎎/㎖)
③ Mix well and incubate at 55∼65℃ for more than 30min.
④ Add 300㎕ of 6M NaCl and vortex 30s at Max. speed.
⑤ Centrifuge for 20min at 12,000rpm
⑥ Transfer supernatant to new tube(700㎕).
⑦ Add 700㎕ of phenol(saturate with TE) and mix well.
⑧ Add 140㎕ of chloroform and mix well.
⑨ Centrifuge 5min at 12,000rpm.
⑩ Transfer supernatant to new tube.
Add equal volume of isopropanol or 2 volumes of EtOH and incubate -20℃ for 30min.
Centrifuge 20min, 4℃ at 12,000rpm or spool out.
Wash with 70% EtOH.
Dry and resuspend with 30∼50㎕ of D.W.
Dissolve all pellet and centrifuge for 3min at 12,000 rpm.
Transfer to new tube the supernatant.
Reagents
Homogenizing buffer for 50㎖

0.4M NaCl NaCl : 1.1688g
10mM Tris-HCl(pH8.0) 1M Tris-HCl(pH8.0) : 0.5㎖
2mM EDTA(pH8.0)
0.5M EDTA(pH8.0) : 0.2㎖
Adjust to 50㎖ with D.W
6M NaCl for 50㎖
Dissolve 17.532g of NaCl to D.W
and adjust to 50㎖ with D.W
20% SDS for 50㎖
Dissolve 10g of SDS to D.W and
adjust to 50㎖ with D.W
DNA Southern blot Analysis
Reagent
Add the 8.62㎖ of HCl(37%) to D.W

0.2M HCl soln.
Adjust volume to 500㎖
0.4N NaOH soln. Dissolve 8g NaOH in D.W
Adjust volume to 500㎖
20 SSC ① NaCl 175.3g
3M NaCl ② Na-citrate 88.2g

0.3M Na-Citrate ③ Dissolve in 800㎖ water, Adjust pH to 7.0 with 10N
NaOH
④ Adjust volume to 1ℓ, Sterilize by autoclaving
Sephadex G-50 ① Wash G-50 10g with 3°D.W (3times)
② Saturate the washed G-50 with TE(pH7.6) (repeat)
③ Discant the supernatant except 160 ㎖ of TE
④ autoclave
50× Denhardt's soln. Ficoll(type4000, Pharmacia) 5g
Polyvinylpyrrolidone 5g
Bovine serum albumin(Pentex, fraction V)
Once dissolved, bring up to 500㎖ total with water
Filter and Store at -20℃ in dark condition
Staining Soln. 2㎍/㎖ EtBr
----------------------
Transfer
① After electrophoresis with positive control in 0.8% agarose gel
Marking the size marker on the vinyl film, take a picture
Pre-hybridization soln (5㎖/bottle) ②

Depurination (large size DNA to transfer efficiently)
Soaking the gel in 0.2M HCl soln for 10min at room temp.
→ bromophenol blue changed yellow color
③ Washing with D.W
④ Denaturation
Soaking in 0.4N NaOH for 30min at room temp.
Pre-soaking NC filter in 0.4N NaOH soln.
⑤ Place the gel on the support in an inverted position.
⑥ Transfer of DNA to proceed for 12∼24hrs, Mark the position of the gel slots on the NC
filter with a pencil.
⑦ Washing NC filter in Water for 10min.
⑧ Soak the filter in 2× SSC for 5min at room temperature.
⑨ Place the filter on a paper towel to dry for at least 15min at room temperature. Bake the
filter for 30min to 2hrs at 80℃ in a vacuum oven or UV cross linking(2000J).
⑩ If the filter is not to be used immediately in hybridization experiments, it should be

wrapped loosely in aluminum foil and stored under vacuum at room temperature.
100㎍/㎖ Salmon sperm DNA 10㎎/㎖ Salmon sperm DNA 50㎕

6× SSC boiling about 18min.(denaturation) → in ice
0.5% SDS 20× SSC 1.5㎖
5× Denhardt's soln 10% SDS 250㎕
50× Denhardt's 500㎕
D.W 2.7㎖ ⇒ final 5㎖
① pre-incubation 65℃ about 10min (or 55℃ 10min)
② Insert membrane into hybridizer bottle and add pre-hybridization soln.
③ pre-hybridization at 65℃ about 1～2hrs. (You can pre-hybridize during overnight)
Hybridizaton
“ The buffer gives reliable results and it is easy to use. For convenience, large volumes of buffer may
be prepared at a time and store at room temp. for at least several months.
This buffer is for every hybridization. Genomic DNA, plasmid, Northern etc. “
Modified Church Buffer from USB by Jae-Hyuk Yu (500㎖)
1 mM EDTA 0.5M EDTA 1㎖

250mM Na2HPO4?7H2O Na2HPO4?7H2O 33.5g
1% Hydrolysated Casein Hydrolysated Casein 5g
7% SDS SDS 35g
85% H3PO4 adjust to pH7.4 ddH2O 400㎖
※ adjust to pH7.4 with H3PO4
Prepare the buffer by adding the reagent in the order listed above. Dissolve the casein
completely before adding the SDS. Adjust volume to 500㎖ with ddH2O. You may
make this buffer as much as 4ℓ and store it at room temperature indefinitely. SDS may
precipitate due low(room) temperature if this happens, don't worry, just heat the buffer
to 68℃ until SDS precipitate disappear and mix well, use it.
----------------------
Probe reaction by random primer extension

① Prepare 25∼100ng double-stranded DNA 10㎕ in EP tube
② Denatured by boiling for 10min
③ Standing on ice for 5min
④ Add :
5× labeling buffer 4㎕
dNTP mixture (-dCTP) 1.5㎕
Klenow (5unit/㎕) 1㎕
32P-dCTP 3㎕ → Adjust volume to 20㎕
⑤ Incubation at 37℃ for 1hr∼2hrs or at room temperature for 4hrs

⑥ Experiment with QIAquick Nucleotide Removal Kit Protocol next step!!
1. Add 10 volume of Buffer PN to 1 volume of the reaction sample and mix

2. Place a QIAquick spin column in a provided 2㎖ collection tube
3. To bind DNA, apply the sample to the QIAquick column and centrifuge
for 1min at 6000rpm.
4. Place the QIAquick column into a clean 2㎖ collection tube and discard the tube
containing the radioactive flow-thorough appropriately.
5. To wash QIAquick column, add 500㎕ of Buffer PE and centrifuge for 1min at
6000rpm
Discard the flow-through appropriately, repeat wash with another 500㎕ of
Buffer PE
6. Discard the flow-through and place the QIAquick column back in the same tube,
which should be empty. Centrifuge for an additional 1min at 12000rpm
7. Place the QIAquick column in a clean EP tube.
8. To elute DNA, add 100∼200㎕ of Buffer EB(10mM Tris-Cl, pH 8.5) or H2O to
the center of the QIAquick membrane and centrifuge the column for 1min at
12000rpm. Alternatively, for increased DNA concentration, add 30∼50㎕
elution buffer to the center of the QIAquick membrane, let the column stand for
1min, and then centrifuge.
⑦ If the radiolabeled probe is double-stranded, denature it by heating for 5min at 100℃.

⑧ Add the denatured probe to the hybridization solution
⑨ Incubate the membrane in bottle set at the appropriate temperature for the required
period of hybridization (usually at 65℃ for 16hrs)
----------------------
Washing
① Discard Hybridization soln

Replace Washing soln A → B → C for 30min, each step twice!
② Place membrane to dry at room temperature.

Place the membrane on the 3MM paper
Cover the membrane with a sheet of wrap
③ Expose the membrane to X-ray film to obtain an autoradiographic

image.Usually be detected after 16∼24 hours of exposure at -70℃ with
an intensifying screen.
Washing solution
A 2× SSC 20× SSC 10㎖

0.1% SDS 10％ SDS 1㎖
DW 89㎖
B 2× SSC 20× SSC 10㎖

1% SDS 10% SDS 10㎖
DW 80㎖
C 0.1× SSC 20× SSC 500㎕

0.1% SDS 10% SDS 1㎖
DW 98.5㎖
----------------------
RNA isolation
with TRI REAGENT by Molecular Research Center. INC.
1. HOMOGENIZATION
- 1㎖ TRI Reagent per 50-100㎎ tissue,10㎠ culture dish or 5-10× 106 cells.
- Store homogenate for 5min.(RT)
2. RNA EXTRACTION
- Add 0.1㎖ bromochloropropane or 0.2㎖ chloroform, mix vigorously.
- Store sample 2-15min.(RT)
- Centrifuge 12000g for 15min.(4℃).
3. RNA PRECIPITATION
- Transfer aqueous phase into a new tube.
- Add 0.5㎖ isopropanol, mix, store for 5-10min.(RT).
- Centrifuge 12000g for 8min.(4-25℃).
4. RNA WASH
- Mix RNA pellet with 1㎖ 75% ethanol.
- Centrifuge 7500g for 5min.(4-25℃).
5. SOLUBILIZATION
- Air dry the RNA pellet for 5-10min.
- Dissolve by pipetting in 50-200㎕ of FORMAzol, 0.5% SDS, or DEPC water and
incubate at 55-60℃ for 10min.
RNA Northern blot analysis

Reagent
5× Formaldehyde ① 3-(N-morpolino)propanesulfonic acid(MOPS) 20.6g

gel-running buffer(MEA) Dissolve in 50mM NaOAc 800㎖
Adjust pH to 7.0 with 2N NaOH
0.1M MOPS (pH7.0) ② Add 10㎖ of 0.5M EDTA
40mM Sodium ③ Adjust volume to 1ℓ with DEPC-water
acetate ④ Filtration with 0.2㎛ filter or sterilize by autoclave
5mM EDTA (pH8.0) ⑤ Store at RT in dark condition
Formaldehyde <100㎖>
gel-loading dye glycerol 50㎖
50% glycerol 0.5M EDTA(pH8.0) 200㎕
1mM EDTA (pH8.0) Bromophenol blue 0.25g
0.25% Bromophenol Xylene cyanol 0.25g
blue
0.25% Xylene cyanol
20× SSC ① NaCl 175.3g
② Na-citrate 88.2g
3M NaCl ③ Dissolve in 800㎖ water, Adjust pH to 7.0 with 10N
0.3M Na-Citrate NaOH
④ Adjust volume to 1ℓ, autoclave.
0.1% DEPC-water. Add 1㎖ of DEPC(diethyl pyrocarborate) in 1ℓ3°D.W
Mix well, incubate at 37℃ for 2hr, autoclave
37% Formaldehyde (12.3M) ① RNase inactivation :cross-linking with RNase,

break disulfide bond
② RNA denaturation : Covalent bond interact with
amine group of A, G, C, prevent to base pair of G-
C, A-T base pair
Formamide Help to Formaldehyde function :
break to hydrogen bond of base
Sephadex G-50 ① Wash G-50 with 3°D.W (3times)
② Saturate the washed G-50 with TE(pH8.0) (repeat)
③ autoclave
50× Denhardt's soln. Ficoll(type4000, Pharmacia) 5g
Polyvinylpyrrolidone 5g
Bovine serum albumin(Pentex, fraction V)
Adjust volume to 500㎖ D.W, filtration
Store at -20℃ in dark condition
Staining Soln. 0.5㎍/㎖ EtBr + 0.1M NH4OAc
Agarose Gel (1.2% total volume 50ml)
① Prepare the gel by melting the 0.6g agarose in 31.1㎖ DEPC-water
② Cooling it to 60℃, adding 5×MEA buffer 10㎖ (final 1 ) and Formaldehyde 8.9㎖ (final
2.2M) to give final concentration, respectively.

Allow the gel to set for at least 30min at room temperature
③ Before loading the samples, pre-run the gel for 5minutes at 5V/cm.
Preparation of RNA reaction mixture
① Prepare the samples by mixing the following in a sterile EP tube:
RNA (up to 30㎍) 4.5㎕ 5 MEA buffer 2.0㎕
Formaldehyde 3.5㎕ Formamide 10.0㎕ 1 : 1.8 : 5 ratio
② Incubate the samples for 15minutes at 65℃, chill them on ice

③ Centrifuge the samples for 5 sec at deposit all of the fluid in the bottom of the EP tube
Gel running
① Add 2㎕ of formaldehyde gel loading dye (1/10volume)
② Fun the gel submerged in 1× formaldehyde gel running buffer at 3-4V/㎝.
③ Soak the gel in DEPC-Water for a minutes, stained with EtBr

④ After de-staining with sterile water, photograph the gel on the UV illuminator
⑤ Transfer the RNA from the gel to a nitro-cellulose membrane
----------------------
Transfer
① Float the NC filter on the surface of 6× SSC for at least 5 minutes

② Fill the dish with 20× SSC until the level of the liquid reaches almost to the
top of the support (such as picture).
③ Allow transfer of RNA to proceed for 6∼18hours. As the paper towels
become wet, they should be replaced.
④ After transfer, mark the positions of the gel slots on the filter with a pencil
⑤ Soak the filter in 6× SSC for 5min at RT
⑥ Place the dried filter between two pieces of 3MM paper, and bake the filter
for 30min to 2hrs at 80℃ in a vacuum oven or UV cross-liking(2000J, 2min).
⑦ If the filter is not to be used immediately in hybridization experiments, it
should be wrapped loosely in aluminum foil and stored under vacuum at
room temperature.
---------------------- Lab of Plant Molecular Biology - Prof. Yang Moon Sik (Korea)
Protein Extraction
- Arakawa et al. (1997)
Expression of cholera toxin B subunit oligomers in transgenic potato plants
1. Tissues (about 1g fresh weight) are homogenized by liquid nitrogen on ice in
1.0㎖ of extraction buffer. Pipetting about 20 times and vortex for 10min.
※ Extraction buffer : 200mM Tris-Cl pH 8.0

100mM NaCl
400mM sucrose
10mM EDTA
14mM 2-mercaptoehtanol
1mM pheylmethylsulfonyl fluoride
0.05% Tween-20
2. The tissues homogenate is centrifuged twice at 15000rpm for 15min at 4℃ to

remove insoluble cell debris.
Protein Determination
- Bradford Assay
Micro Assay (Range 1-10㎍ of protein per assay)
1. Gently mix the dye reagent in the bottle.
2. Protein standard protein solutions ranging from 1㎍/㎖ to 10㎍/㎖ using bovine
serum albumin or equivalent protein standard solution. To obtain best results, make
serial dilutions from the highest standard concentration.
3. Add 1㎖ of the standard protein solution to each appropriately labeled tube. In the
tubes labeled blank, add 1㎖ of distilled water.
4. Prepare the unknown samples as above with approximate concentration between

1㎍/㎖ to 10㎍/㎖. Aliquot 1㎖ of unknowns to each tube. Prepare all standards
and samples in duplicate.
5. To each tube, add 1㎖ of Bradford Reagent and mix.
6. Prior to reading absorbance, blank the spectrophotometer with distilled water.
7. Read absorbance between 5-60min at 595nm using disposible cuvet.

The Protein-dye complex is stable up to 60 mins.
8. Substrate the A595 of the blank from the A595 of the standard solutions and
unknown samples
9. Plo; the average absorbance vs. concentration of the duplicate standard protein
solutions,
10. Determine the protein concentration of the unknown samples by comparing the
A595 values against the standard curve.
References:
1. Braford MM : A refined and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal. Biochem. 72.248(1976)
2. Semak JJ, Grossberg SE : A rapid, sensitive and versatile assay for protein using Coomassie brilliant
blue G250. Anal. Biochem. 79.544(1977)
Protein Western blot analysis
Reagent
30 % acrylamide solution ① 29g acrylamide 1g

N,N'-methylenebisacrylamide
Dissolve in 60㎖ DW, Adjust volume to 100㎖
② After filtration store at 4℃ in dark bottle
4× separating gel buffer ① 2M Tris 75㎖ + 10% SDS 4㎖
1.5M Tris (pH8.8) ② Adjust volume to 100㎖ with DDW

0.4 % SDS (stable for months at 4℃)
4× stacking gel buffer ① 1M Tris 50㎖ + 10% SDS 4㎖
0.5 M Tris (pH6.8) ② Adjust volume to 100㎖ with DDW

0.4 % SDS (stable for months at 4℃)
10 % ammonium Dissolve with DDW
persulfate.
2× SDS sample buffer ① 1M Tris 12.5㎖
② SDS : 4g
0.125 M Tris-Cl (pH 6.8)
4% SDS ③ glycerol 20㎖
20% glycerol ④ 2-mercaptoethanol : 10㎖
10% 2-mercaptoethanol ⑤ bromophenol blue : 0.2g
0.2% bromophenol blue Dissolve and bring to 100㎖ with water
Store at room temperature
(stable for week in the refrigerator or for
months at -20 ℃)
Electrophoresis buffer ① Tris 3g + glycine 14.4g + SDS 1g
② Adjust volume to 1ℓ DDW
0.025 M Tris Adjust pH to 8.3～8.8 with HCl
0.192 M glycine
0.1 % SDS
Gel staining solution ① coomassie Brilliant Blue R250 : 2.5 g
(1ℓ) ② methanol : 450㎖
③ H2O : 450㎖
④ glacial acetic acid : 100㎖
⑤ Filter the solution through a Whatman No.1 filter
in order to remove some particle matters.
Continue to next page….
Gel destaning solution ① methanol : 300㎖
(1ℓ) ② glacial acetic acid : 100㎖

③ H2O : 600㎖
Transfer buffer (pH8.3) ① Tris : 5.8g
(1ℓ) ② glycine : 2.9g
③ SDS : 0.37g
48mM Tris ④ methanol : 200㎖
39mM glycine
0.037% SDS
20% methanol
or ① Tris : 3.03g
② glycine : 14.4g
25mM Tris ③ methanol : 200㎖
192mM glycine
The buffer will range from pH8.1 to 8.4
20% methanol
Blocking buffer (500㎖) ① skin milk : 25g
② Tween 20 : 50㎕
1～5% skin milk ③ 5% Sodium azide : 2㎖ (option)
0.01 % Tween 20 ④ dissolve in PBS or TBST
(option) 0.02 % Sodium
azide
PBS buffer (pH7.4) ① NaCl : 8g
② KCl : 0.2g
③ Na2HPO4 : 1.44g (Na2HPO4◦12H2O 3.5814g)
④ KH2PO4 : 0.24g
⑤ Adjust pH with NaOH and bring to 1ℓ with water
TBST (500㎖) ① 1M Tris(pH7.5) : 50㎖
② NaCl : 4.5g
100mM Tris(pH7.5) ③ Tween 20 : 500㎕
0.9% NaCl
0.1%Tween 20
TMN (500㎖) ① 1M Tris(pH 9.5) : 50㎖
② 5M NaCl : 10㎖
100mM Tris(pH9.5)
100mM NaCl ③ 1M MgCl2 : 2.5㎖
5mM MgCl2
----------------------
Make the SDS-polyacrylamide Gel
① Assembly the Vertical slab gel unit

X % Seperating Gel
② Pour the acrylamide solution into the gap between acrylamide stock soln. : X/3 ㎖
the glass plates. Leave sufficient space for the stacking 4× separating gel buf. : 2.5 ㎖
gel (the length of the teeth of the comb plus 1㎝). H2O : (7.5 - X/3)㎖
10% APS : 50㎕
③ Using a pasteur pipette, carefully overlay the 0.3㎖ TEMED : 5㎕

( X <8% : 10㎕)
water-saturated n-butanol or DDW
④ Place the gel at RT for 30min
⑤ Pour off the overlay and wash the top of the gel several times with DW
⑥ Remove any remaining water with filter paper
⑦ Pour the stacking gel solution directly onto the surface of the polymerized resolving gel.
Immediately insert a clean Teflon comb into the stacking gel solution.
⑧ Polymerization is complete (30minutes)
Stacking Gel (5 %)
Recommended % Protein size
of acrylamide range acrylamide stock soln. : 0.67㎖
8% 40 ∼ 200 KDa 4× stacking gel buf. : 1.0㎖
10 % 21 ∼ 100 KDa
12 % H2O : 2.3㎖
10∼ 40 KDa
10% APS : 30㎕
TEMED : 5㎕
Pouring the SDS-Polyacrylamide gel
Prepare the samples by heating them to 100℃ for 3minutes in 1× SDS sample buffer
to denature the proteins
----------------------
Gel electrophoresis
① After polymerization is complete (30minutes), remove the Teflon comb carefully.

② Using a squirt bottle, wash the wells immediately with DW to remove any unpolymerized
acrylamide.
③ Mount the gel in the electrophoresis apparatus.
④ Add Tris-glycine electrophoresis buffer to the top and bottom reservoirs.
⑤ After the sample loading, run the gel until the bromophenol blue dye reaches the bottom
of the resolving gel.
Stacking gel : 50V, Separating gel : 100V
⑥ The gel stained with Coomassie Brilliant Blue.
Transfer
① When the electrophoresis is finished, soak the gel in transfer buffer for 10∼15min.
② Place the gel onto nitro-cellulose filter, set up such as picture.
Pour the transfer buffer in electro-transfer tank.
● Wearing gloves, set up the transfer apparatus, gel,

3MM paper, NC filter
● Soak 3MM paper, nitrocellulose filter in transfer buffer
● Select NC filter with appropriate pore size depend
on the protein size
( ≥15KDa : 0.45㎛, ≤15KDa :0.2㎛)
● To transfer efficiently use circulation system. When the
temperature increased, forming air bubble in gel
sandwich
Continue to next page…………..
③ Apply a current 250mA ∼ 300mA for 1hr

※ 210mA, 60V for 5hr or 100mA, 30V overnight transfer
④ Take out the filter, add 0.1㎖ of blodking buffer per square centimeter of filter
Incubate the filter for 1∼2 hours at room temperature with gentle agitation on a platform
shaker (or overnight at 4℃)
※ blocking solutes : 5% skin milk, 3% BSA, 2.5% gelatin, 2% casein
⑤ Discard the blocking buffer and Washing the filter 3 times with PBS buffer
⑥ Add an appropriate quantitiy of the primary antibody
Incubate the filter for 2 hours at 4℃ with gentle agitation on a platform shaker
※ The appropriate amount of primary antibody should be determined empirically in pilot
experiments. The recommended test dilutions are :
polyclonal antibody : 1/100 - 1/5000

supernatant from cultured hybridoma cell : 1/10 - 1/100
ascitic fluid from mice being hybrid myelomas : 1/1000 to 1/10,000
⑦ Discard the antibody buffer and washing the filter 3 times with PBS buffer
⑧ Discard PBS buffer and the add the blocking buffer(or TBST) containing the secondary
antibody be diluted 1/1000 to 1/7000 for 2 hour at room temperature with gentle agitation
⑨ Wash the NC filter with TBST 3 times for 5min and then with TMN 1 times for 5min
⑩ For washing, prepare the 33㎕ of NBP and 16.5㎕ of BCIP in 5㎖ TMN solution
(developing solution)
Pour the developing solution, Monitor the progress of the reaction carefully
NBT 50㎎/㎖ in 70% DMF

(Nitro blue tetrazolium)
BCIP 50㎎/㎖ in DDW
(5-Bromo-4-chloro-3-indolyl phosphate)
⑪ After develop wash the NC filter with DDW, store on the3MM paper in dark condition
----------------------
Cyotokine ELISA Protocol
● Capture antibody
1) Dilute the purified anti-cytokine capture antibody to 1―4 ㎍/㎖ in Coating buffer
Add 50㎕ of diluted antibody to the wells of the ELISA plate
(e.g., Nunc Maxisorb; Cat. #2442404)
2) Seal the plate to prevent evaporation. Incubate overnight at 4℃
● Blocking
3) Bring the plate to room temperature, remove the capture antibody solution,
and block non-specific binding by adding 200㎕ of Blocking Buffer per well.
4) Seal plate and incubate at room temperature for 1-2hours.
5) Wash ≥3 times with PBST
● Standards and Samples

6) Add standards and samples (diluted in Blocking Buffer/Tween) at 100㎕ per well
7) Seal the plate and incubate it for 2-4 hours at room temperature or overnight at 4℃ 8)
Wash ≥ 4 times with PBS/Tween
● Detection antibody
9) Dilute the biotinylated anti-cytokine detection antibody to 0.5-2㎍/㎖ in Blocking
Buffer/Tween. Add 100㎕ of diluted antibody to each well
10) Seal the plate and incubate it for 1 hours at room temperature
11) Wash ≥ 4 times with PBS/Tween
● Avidin-Horseradish Peroxidase(HRP)
12) Dilute the avidin- or streptoavidin-HRP conjugate or other enzyme conjugate to
its pre-titered optimal concentration in Blocking Buffer/Tween. Add 100㎕ per
well.
13) Seal the plated and incubate it at room temperature for 30 minutes.
● Substrate
15) Thaw ABTS substrate within 20 minutes of use. Add 100㎕ of 3% H2O2 per 11㎖ of
substrate and vortex. Immediately dispense 100㎕ into each well. Incubate at room
temperature (5-80 minutes) for color development. The color reaction can be stopped by
adding 50㎕ of Stopping Solution
16) Read the optical density (O.D.) for each well with a microplate reader set to 405
nm.
GM1 Binding Assay
● Capture antibody
1) Dilute the purified GM1-ganglioside cell to 3 ㎍/㎖ in Coating buffer

Add 100㎕ of GM1-ganglioside cell to the wells of the ELISA plate
And 100㎕ of BSA(3 ㎍/㎖) as control.
(e.g., Nunc Maxisorb; Cat. #2442404)
2) Seal the plate to prevent evaporation. Incubate overnight at 4℃
● Blocking
3) Bring the plate to room temperature, remove the remained proteins solution,
and block non-specific binding by adding 300㎕ of Blocking Buffer per well.
4) Seal plate and incubate at 37℃ for 1-2hours.
5) Wash ≥3 times with PBST
● Standards and Samples

6) Add standards and samples (diluted in Blocking Buffer/Tween) at 100㎕ per well
7) Seal the plate and incubate it for 2-4 hours at 37℃ or overnight at 4℃
● Detection antibody
9)Dilute the Rabbit anti-LT-B (Sherwood R97140) 1:5000 in Blocking
Buffer/Tween. Add 100㎕ of diluted antibody to each well
10) Seal the plate and incubate it for 1 hours at room temperature
● Avidin-Horseradish Peroxidase(HRP) (1:10000)

12) Dilute the anti-rabbit IgG antibody streptoavidin-HRP conjugate
to its pre-titered optimal concentration in Blocking Buffer/Tween.
Add 100㎕ per well.
13) Seal the plated and incubate it at room temperature for 30 minutes.
● Substrate
15) Thaw ABTS substrate within 20 minutes of use. Add 100㎕ of 3% H2O2 per 11㎖ of
substrate and vortex. Immediately dispense 100㎕ into each well. Incubate at room
temperature (5-80 minutes) for color development. The color reaction can be stopped
by adding 50㎕ of Stopping Solution
16) Read the optical density (O.D.) for each well with a microplate reader set to 405
nm.
----------------------
♥ Solutions
Coating Solution ( for cytokine)

NaHCO3 8.4 g
Na2CO3 3.56 g
Adjust volume to 1L D.W. and adjust pH 9.5 with 0.1M NaH2PO4
Coating Solution ( for GM1 binding )

NaHCO3 8.4 g
Na2CO3 3.56 g
Adjust volume to 1L D.W. and adjust pH 9.5 with 0.1M NaH2PO4
PBS Solution
80.0 g NaCl
11.6 g Na2HPO4
2.0 g KH2PO4
2.0 g KCl Adjust volume to 1ℓ, pH to 7.0
PBS/Tween
0.5㎖ of Tween-20 in 1ℓ PBS
Blocking Buffer
Prepare 10% fetal bovine serum(FBS), 10% newborn calf serum(NBCS) or 1% BSA
(immunoassay grade) in PBS. The Blocking Buffer should be filtered to remove paticulates
before use.
Blocking Buffer/Tween
Add 0.5㎖ Tween-20 to 1ℓ Blocking Buffer
ABTS Substrate Solution

Add 150㎎ 2,2'-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (e.g., Sigma, Cat.
#A-1888) to 500㎖ of 0.1M anhydrous citric acid (e.g., Fisher; Cat. #A-940) in
ddH2O; pH to 4.35 with NaOH. Aliquot 11㎖ per vial and store at -20℃. Add 100㎕ 3%
H2O2 prior use.
3% H2O2 Solution
Add 10㎖ of 30% H2O2 to 90㎖ of H2O2. Protect from prolonged exposure to light
Stopping Solution (20% SDS/50% DMF)

Add 50 ㎖ of dimethylformamide (DMF) to 50㎖ ddH2O then add 20.0g sodium dodecyl
sulfate(SDS) or H3PO4 34.1 ml, up to 500ml D.W.

Protocol English PMB Lab Prof. Yang

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Protocol of PMB Lab - Chonbuk National University

Preparation of Competent E.coli - Ⅱ

1. Inoculate 5㎖ LB medium with single colony and overnight culture

3. Grow up it until OD@600 = 0.5 (200rpm, 1.5∼3 hrs) at 37℃.

7. Spin 4000rpm for 5 min at 4℃.

1. Throw competent cell in ice.

2. Aliquot 10㎕ of ligation mixture to a new tube.

3. Add 200㎕ of competent cell into a ligation mixture.

4. Inoculation in ice for 30min ∼ 1hr with intermittent tapping.

5. Heat shock at 42℃ for 90sec.

6. Put the tube into ice for 3mins.

8. Spreading onto plate (containing your favorite antibiotics).

10. Invert the plate and incubate at 37℃ for 12 ∼ 15hrs.

One step miniprep method

1. Inoculation of transformant in a EP tube (LB + antibiotics, 0.5㎖).

2. Add 0.5㎖ of phenol:chloroform:isoamylalcohol(25:24:1).

3. Mix by vortex at the maximum speed for 1min.

4. Spin at 12000rpm for 5min.

6. Add 0.4㎖ of the isopropanol and mix well.

7. Immediately, spin at 12000rpm for 5min.

9. Vacuum dry the pellet.

10. Suspend in 20㎕ of TER(TE+20㎍ RNAse/㎖).

11. About 5㎕ of this DNA can be cleaved with appropriate restriction

enzymes for analysis.

Mini-Prep of plasmid DNA (Alkaline lysis method)

1. Inoculate 5 ml culture of the proper medium (ie. with antibiotics) from a

2. Transfer 1.5ml EP tube, spin 15sec.. Decant the supernatant.

6. Centrifuge at 12000rpm for 10min.

7. Transfer the supernatant into the new EP tube,

10. Vacuum drying the DNA pellet.

Elution of DNA fragment using Geneclean kit (from Bio101)

1. Excise gel slice and place it in a EP tube

2. Add 3 volume of sodium iodide (100㎕=0.1g agarose)

3. Heat in a 45∼55℃ water bath for 5min to melt gel.

5. Centrifuge at 12000rpm for 20sec, discard supernatant.

6. Add 500㎕ of Washing buffer, suspend by pipetting back and forth)

7. Add any volume of TE or DW 20㎕, vortex softly

8. Heat in a 45∼55℃ water bath for 5min

9. Centrifuge at 12000rpm for 1min

1. Grow the cell in 5㎖ LB media with vigorous shaking at appropriate

A. tumerfaciens (100㎍/㎖ Rifampicine, 200㎍/㎖ Sm)

2. Mix the three bacteria!!

3. Select the bacteria using the antibiotics

④ Spread the 100㎕ of diluted sample on the LB plate

1. Grow Agrobacterium cell in 5㎖ YEP(or LB) with vigorous shaking at

28℃. (add appropriate antibiotics and culture overnight)

incubate for at least 30min at room temperature.

5. Add 30㎕ phenol(equilibrated with 2 volume of SolⅡ) and vortex gently

for a few sec.

6. Add 150㎕ of 3M NaOAc(pH4.8) and shake the tube briefly.

7. Store the tube at -20℃ for 15min.

12. Add 500㎕ of 0.3M NaOAc(pH7.0) and 2 volume of 95% EtOH.

Store at -70℃ for 15min.

15. Dissolve pellet in appropriate TE buffer.

1. Use mass to make leaf disks. size: 0.5㎠

2. Soaking leaf disks in the MS104 medium

4. Inoculate Agrobacterium in the soaking leaf disks for 10min

5. Transfer to sterilized filter paper over MS104 plate.

6. Culture at 25℃ for 2∼3days in dark condition