MG Lab Protocols

8/16/2019 MG Lab Protocols

1/40

DNA gel purification (UltraCleanTM 15 Kit):

1. Cut the gel band under UV light (2 wells for 50 ul digestion).

2. Add 1 ml of ULTRA SALT and melt the gel at 55-65 C for 10 min (mix occasionally to

make sure the gel melt completely).

3. Add 6 ul of ULTRA BIND (resuspend thoroughly by vortexing), and mix well. Incubate for10 min at RT (mix several times).

4. CF 15 seconds and remove supernatant.

5. Resuspend the pellet in 1 ml of ULTRA WASH.

6. CF 15 seconds and remove supernatant. Wash with ULTRA WASH one more time.

7. Vacuum dry the pellet for 3 min.

8. Resuspend the pellet in 30 ul of H2O or TE (disturb the pellet with the pipette tip to

resuspend).

9. CF 1 min and remove the supernatant (DNA in the supernatant).

Ligation:5X Buffer 2 ul

Insert (gel purified) 3 ul

Vector (gel purified) 1 ul

10 mM ATP 0.5 ul

T4 Ligase 1 ul

H2O 2.5 ul

Mix well and keep at RT 1 h-o/n.

Transformation and plating:1. Thaw competent cells on ice

2. Mix 5 ul of the ligation reaction and 50 ul of cells in 1.5 microfuge tube.

3. Incubate on ice for 20-30 min.

4. Heat-shock the cells at 42 C for 30-45 seconds.

5. Place the cells on ice.

6. Add 500 ml of LB medium to the cell and shake the tube at 37 C for 45 min to1 h.

7. Spread 150 ul culture onto LB plate containing proper antibiotics. Incubate o/n

at 37 C.

Or Pellet cells at 5000 rpm for 1 min and quickly pour off the LB (about 100-150 ul

left-over LB + pellet still in the tube). Resuspend the pellet and plate entire

contents ont LB + proper antibiotics plates.


2/40

TOPO TA Cloning (kit):

PCR reaction:

Buffer (with Mg) 5 ul

DNA (template) 0.5 ul of Miniprep DNA (100-500 ug)

dNTPs (10 mM) 1 ul

3’-primer (50 pmoles/ul) 0.5 ul

5’-primer (50 pmoles/ul) 0.5 ul

Enzyme (DyNAzyme) 0.5 ul

H2O 42 ul

Add 2-3 drops of mineral oil, use hot start. Heat up to 95 C for 1 min, then go to:

94 C 30’’

55 C 30’’ (Tm of primers should be > 60 C, if not lower annealing T)72 C 1’ (for long fragment, increase 1’/kb)

n=35

Link to 72 C---10’, then keep at 4 C. Take 2-5 ul run gel.

TOPO ligation (kit):

PCR product 2 ul

Salt Solution 1 ul

TOPO vector 0.5 ul

H2O 1.5 ul

Mix and incubate for 15 min at RT, then place the tube on ice.

Transformation and plating (kit):

1. Thaw One Shot E. coli on ice.

2. Add 2 ul of the ligation reaction to 50 ul competent cell and gently mix.

3. Incubate on ice for 15 min.

4. Heat-shock the cells at 42 C for 30 seconds.

5. Place the cells on ice.

6. Add 250 ul of RT SOC medium to the vial, and shake the tube at 37 C for 1 h.

7. Spread 100 ul culture onto LB plate containing antibiotics and/or X-Gal (spread 50 ul of

20 mg/ml on the plate and let it dry before spread the cells).

8. Incubate o/n at 37 C.

9. Pick white or light blue colonies (if use X-Gal) for Mini-prep.


3/40

Mini-Prep (alkaline lysis):

1. Pour 1.5 ml of an o/n culture into a microtube, CF for 1 min and discard the supernatant.

2. Resuspend the cells in 100 ul of Solution 1 by vortexing.

3. Add 200 ul of Solution 2, mix by inverting the tubes twice and incubate for 2 min at room T.

4. Add 150 ul of cold Solution 3, mix by inverting twice and incubate for 5 min on ice.

5. CF for 5 min and carefully pipette the supernatant (380 ul) in a new tube.

6. Add 1 ml 100% ethanol, invert twice and CF for 2 min, discard the liquid, wash the pellet

with 70% ethanol and vacuum dry the pellet for 5 min.

7. Resuspend the final product in 50 ul TE with RNase A (20 ug/ml).

8. Take 2 ul to do enzyme digestion.

Solution I (4 C): 50 mM glucose in 50 mM Tris-HCl pH 8.0, 10 mM EDTA, or in regular TE

(10 mM Tris-HCl, 1 mM EDTA, pH 7.5-8.0).

Solution II (RT): 0.2 N NaOH, 1% SDS.

Solution III (4 C): 3 M potassium acetate adjusted to pH 5.5 with glacial acetic acid (100 ml

Solution III: 60 ml 5 MKAc, 11.5 ml glacial HAc, 28.5 ml H2O).

Note: After step 5, can directly load 15 ul/well (no need for loading buffer, nor ladder either)onto a gel and run for 25 min at 150 V to check if we got insert (the bigger plasmid). This is

good for large number selection.

Enzyme digestion (for Mini or Midi preps):

Plasmid DNA 2 ul

10X buffer 1 ul (make sure to use the right buffer for the enzyme)

Enzyme 0.2 ul

H2O 6.8 ul (to make up total volume of 10 ul)

Incubate at 37 C (some enzyme needs special T) for 30 min. Load total digestion on the

gel to check if you can get the right gel band (good luck).


4/40

Mini-Prep (lysozyme-boiling lysis):

1. Pour 1.5 ml of an o/n culture into a microtube, CF for 1 min and discard the supernatant

completely.

2. Resuspend the cells in 200 ul of STET-lysozyme (mix 180 ul of STET and 20 ul of

lysozyme before use) by vortexing and keep at RT for 5 min.

3. Place the tubes in boiling water for 1 min.

4. CF for 10 min at max speed.

5. Remove the cell debris with a toothpick.6. Take 8 ul of supernatant to run the gel to check it contains insert or not.

For those plasmids with insert:

7. Add H2O to make up to 200 ul, then add 200 ul of isopropanol and mix well by inverting

tubes several times.

8. CF for 10 min at max speed, wash the pellet with 70% EtOH, dry the pellet and

resuspend in 50 ul TE + RNase.

9. Take 2 ul to do enzyme digestion.

Solutions (store at 4 C separately):

STET: (10% sucrose, 50 mM Tris-HCl, pH 8.0, 50 mM EDTA, 0.2% Triton X-100).Lysozyme: 20 mg/ml in STET.


5/40

Midi-Prep (Bio-RAD kit):

1. Inoculate plasmid-containing bacteria into 40 ml of LB containing proper antibiotics in a

flask. Culture o/n at 37 C in a rotary shaker.

2. CF for 5 min in a tube with cap, and discard the supernatant.

3. Add 5 ml of Cell Resuspension Solution and vortex to resuspend the pellet.

4. Add 5 ml of Cell Lysis Solution and invert twice (do not vortex).5. Add 5 ml of Neutralization Solution, invert twice and sit on ice for 5 min.

6. CF for 10 min at 10,000 rpm and carefully pour the supernatant to a new tube.

7. Add 800 ul of resuspended Quantum Prep Matrix and mix well. Keep the tube at RT for

10 min (mix from time to time).

8. CF for 2 min at 8000 rpm and pour off the supernatant. Add 10 ml of Washing Buffer and

resuspend the matrix.

9. CF for 2 min at 8000 rpm and pour off the supernatant. Add 10 ml of Washing Buffer and

resuspend the matrix. CF for 2 min at 8000 rpm and pour off the supernatant.

10. Add 500 ul of Washing buffer and resuspend the matrix.

11. Transfer the matrix into a spin column with the tip snapped off sitting inside a 2 ml collection

tube. Puncture a hole in the column cap and CF for 1 min at 10,000 rpm.

12. Discard the buffer from the tube and put the column back to the tube. Add 500 of Washing

buffer and CF for 1 min at 10,000 rpm.

13. Discard the buffer from the tube, put the column back to the tube and CF for 2 min at

maximum speed.

14. Transfer the column to a clean tube, add 400 ul of H20 or TE to the matrix, sit for 5 min, and

CF for 2 min at maximum speed.

15. Take 2 ul to do enzyme digestion and run the gel to check the concentration.

Note: Once in a while, plasmid can be lost during o/n culture. To make sure you have the

plasmid, you can take 1 ml of o/n culture to do quick Mini-prep and run a quick gel (withoutenzyme digestion) to check the concentration.

After step 14, you should add another 200 ul of H20 or TE to the matrix and save the column. If

your final concentration too high, you can spin down this and combine both elutions.


6/40

Agro-Infiltration:

1. Grow A.t on plate containing proper antibiotics for 2 days at 28 C.

2. Scrape the cells into 4 ml of 10 mM MES with 10 mM MgCl2.

3. Vertex to resuspend cells, and take 1 ml to check OD600.4. Adjust the left-over 3 mls to 0.6 (OD600) with above MES.

5. Add 0.1 M acetosyringone (1.5 ul/ml), mix and sit in RT for 3 h.

6. Use a syringe to infiltrate the cell suspension from the under side of the leaf (2-3 leaves/plant).

7. Mark the edge of the infiltrated area and keep the plant in a growth room.

Note: dissolve acetosyringone in DMSO

Making competent cells of Agrobacterium tumefaciens:

1. Inoculate 6 ml LB Rif100 with 15 ul glycerol stock and overnight shaking at 28 C.

2. Inoculate 50 ml LB Rif100 in 250 ml flask with 5 ml of overnight culture.

3. Grow culture at 28 C for about 8 h to an OD600 of 0.6.

4. Chill culture on ice for 5 min. Pellet cells at 4000 rpm for 5 min. Resuspend in 1 ml 20 mM CaCl2.

5. Make 100 ul aliquots and store at -80 C. Use 25-50 ul of competent cells for each transformation.

Transformation of Agrobacterium tumefaciens:

1. Add 5 ul purified plasmid to 25 ul of competent cells in a 1.5 ml tube and mix by gently tapping the

tube. Freeze cells in liquid nitrogen.

2. Thaw frozen cells in 37 C water bath for 5 min and then add 800 ul of LB (no antibiotics).


7/40

3. Incubate cells with shaking at 28 C for 2-3 h. Pellet cells at 5000 rpm for 40 sec and quickly pour off

the LB (about 100-150 ul left-over LB + pellet still in the tube). Resuspend the pellet and plate entire

contents onto LB + proper antibiotics plates. Put the plates to 28 C.

4. 2-3 days you should see colonies.

Antibiotics stock solution

Rifampicin: 25 mg/ml in 100% methanol

Kanamycin: 25 mg/ml in water

Tetracycline: 5 mg/ml in 100% ethanol

Spectinomycin: 50 mg/ml in water

Competent Cells (NEB M13 sequencing manual)

1. Inoculate 7 ml of LB with one colony from a plate and incubate at 37 C overnight

with shaking (225 rpm).

2. Add 4-6 ml of overnight culture into 200 ml fresh media and shake at 37 C until

OD600 is 0.4-0.6 (approximately 2-3 h).

3. Chill on ice briefly. Keep everything at 4 C from this point on.

4. Pellet cells at 5000 rpm for 5 min at 4 C.

5. Gently resuspend cells in 40 ml (1/5 of the original culture volume) of ice-cold 0.1 M

MgCl2. Keep on ice for 15-20 min.

6. Centrifuge at 4000 rpm for 5 min at 4 C.

7. Gently resuspend cells in 4 ml (1/50 of the original culture volume) of ice-cold 0.1 M

CaCl2.

8. Keep on ice 1-2 h to establish competency. The cells may be frozen at this point by

adding 1 ml (1/4 volume) of sterile 75% glycerol and placed at –80 in 0.1 ml aliquots.

When retrieving cells, thaw on ice and use immediately.

SYNV Inoculation:

Grind tissue in 0.01 M Na-PB, pH6.8-7.0 containing 1% celite and 0.5% Na2SO3.

Note: weigh celite and Na2SO3, mix with the buffer before inoculation.


8/40

Protein Concentration Assay (Bradford: Bio-Rad):

Prepare standard IgG: our stock is 1.43 mg/ml

Concentration 0.2 0.4 0.6 0.8 1.0 1.2 mg/mlOur stock 14 31 42 56 70 84 ul

H2O 86 69 58 44 30 16 ul

Add H2O first to 13 mm glass tube, then add IgG stock.

Dilute dye solution: 8 ml dye + 32 ml H2O (dye:water = 1:4)

Prepare your sample: 1 to 10 dilution:10 ul + 90 ul H2O or 1 to 5 dilution: 20 ul + 80 ul H 2O

Color reaction: Add 5 ml diluted dye into each tube and incubate at room temperature for more

than 5 min.

Measurement: Visually check your sample to see what concentration range it should be. Then

take three surrounding standard concentrations plus your sample to measure absorbance at 595

nm (600 nm ok too). Use your diluted dye solution to zero the machine.


9/40

Preparation of TNM-FH insect medium

1. Get ~800 ml of water and start stirring. Add one bottle of the powdered medium and

keep stirring until dissolved (do not heat).

2. Add 0.35 g of sodium bicarbonate and adjust the pH to 6.2 with NaOH.

3. Add 100 ml of FBS and more water to make up to 1 L.

4. Filter through 0.45 um filter (need change filter a few times, use big red tube for

vacuum from bench).

5. Filter through 0.22 um filter (under sterile condition: screw the filter to an autoclaved

bottle under hood, then filtering on the bench with the vacuum).

6. Aliquot into 50 ml sterile plastic tube under hood and store at 4 C.

Typhoon Scanner Usage (pw: plantpath)


10/40

1. Wipe the glass window and put your Phosphor screen face down at the corner of A and 12. Doule clic! Typhoon Scanner icon". Select area (#ed corresponding to our Phosphor screen) you want to scan$ then clic! Scan

(sa%e your file into your folder)&. 'pen your file with age*uant (this progra is open all the tie)

+. ,lic! -dit age icon and a!e adustent/. ,lic! 0ileSa%e s and sa%e your file as tif file into your flash dri%e

SDS-GEL ELECTROPHORESIS: (from RY)

Assembly of gel apparatus

Casting the gel

1. Prepare the separating gel solution by combining all reagents (add APS and TEMED last) and pour t

solution to the gel plate (the top of the gel will be 1 cm below the teeth of the comb; if use 0.75 space

pour 3.5 ml of gel solution).

2. Immediately overlay the gel solution with water or H2O saturated isobutanol.

3. Allow the gel to polymerize for 45 min to 1 h (you can see clear 2 lines between solutions).

4. Prepare the stacking gel solution by combining all reagents except APS and TEMED.

5. Rinse off the overlay solution completely with water. Blot-dry the area above the separating gel wi

filter paper.

6. Add APS and TEMED to the stacking gel solution, mix and pour the solution into the gel plates. Pla

the comb in the gel sandwich.


11/40

7. Allow the gel to polymerize for 30-45 min. Remove the comb and rinse the sample wells complete

with water.

Sample treatment

Sample from infected plant: Use 1.5 ml centrifuge tube lid to cut a leaf disc, add 200 ul of 2X samp

buffer, grind the disc and place in a boiling water bath for 10 min. Centrifuge for 5 min and take tsupernatant to load on the gel. This sample can be stored at 4 C or RT for long time (do not need to bo

before loading).

Assemble gel apparatus (be sure to follow manufacture’s instructions to avoid leaking)

After assemble the gel apparatus, add electrode buffer (1X).

Loading sample

Load 10-20 ul of sample to each well. Load 5 ul of SDS-GEL standard (unstained for Coomassie a

prestained for Weste rn; do not need to boil, just thaw and load).

Running the gel

Connect the electric ware and run the gel for ~1 h at 175 V (let dye reach the gel bottom).

Remove the gel

Disassemble the gel apparatus, open the gel sandwich and remove the gel carefully.

Staining

Follow PageBlue staining protocol.

Separating Gel (8 ml for 2 gels when use 0.75 mm spacer, 3.5 ml/gel)

15% 12% 10% 7.5%

Water (ml) 1.86 2.68 3.34 3.881.5 M Tris-HCl, pH 8.8 (ml) 2.0 2.0 2.0 2.0

10% SDS 80 ul 80 ul 80 ul 80 ul

Acrylamide/Bis (ml) 4.0 3.2 2.66 2.0

(30% stock, 37.5:1)

10% APS 80 ul 80 ul 80 ul 80 ul

TEMED 4 ul 4 ul 4 ul 4 ul

***For APS: Prepare fresh and good for 1 wk at room C, or 1 month at 4 C***

Stacking Gel (3 ml for 2 gels) 4%

Water 1.8 ml

0.5 M Tris-HCl, pH 6.8 0.75 ml

10% SDS 40 ul

Acrylamide/Bis (30% stock) 0.39 ml


12/40

10% ammonium persulfate (APS) 60 ul

TEMED 3 ul

Running Buffer (5X stock)Tris base 15 g

Glycine 72 g

SDS 5 g

Make to 1000 ml with water.

2X Sample buffer (total 10 ml)

Water 3.5 ml

0.5 M Tris-HCl, pH 6.8 2 ml (pH range should be 6.8-7.5)

Glycerol 2 ml

10% SDS 2 ml

ß-mercaptoethanol 0.5 ml (add before use)

bromophenol blue 0.01 g

Sample Concentration

Purified virus ~5 ug/lane

Protein ~5 ug/lane

WESTERN BLOT:

1. Soak all transfer cassette parts (fiber pad, 3MM filter paper, nitrocellulose paper) in transfer buffer.

2. Assemble in the following order onto the black side (current: black--->white) of gel cassette.

a. Fiber pad

b. 3MM filter paper

c. Gel

d. Nitrocellulose papere. 3MM filter paper

f. Fiber pad

3. Keep the black side of cassette on the black side of the transfer apparatus.

4. Blot at 0.25A for 1.5~2 h (with cold transfer buffer, in a container and stirring).

5. Block non-specific sites with 15-20 ml of 5% dry milk in TBS for 30 min on shaker.


13/40

6. Add primary Ab (1:1000-4000 or 2~5 ug/ml) into the blocking buffer. Incubate overnight on slo

shaker.

7. Wash 3 X 10 min in H2O on shaker.

8. Incubate the membrane with secondary Ab (make sure use right Ab at proper dilution) in blockin

buffer. Incubate 2 h on slow shaker.

9. Wash 3 X 10 min in H2O on shaker. Ready to do Chemiluninescent exposure (step 12) or Col

development (step 10).

10. Develop in the dye solution (cover from light for about 10 min).

11. Rinse with water and air dry the membrane.

12. Drain the membrane by touching a corner on a paper towel, then place it on plastic wrap on a fl

surface (do not let the membrane dry).

13. Pipette a thin layer of substrate solution (CDP-Star Ready-to-use, 1-2 ml) onto the blot and incubate f

5 min.

14. Drain the substrate solution and then wrap the membrane in plastic wrap.

15. Place a standard X-ray film over the membrane and expose for 10 sec-5 min. Develop the film.

Dye solution: (For each blot, add 50 ul of each dye into 12 ml of buffer, pH9.5)NBT (Nitri Blue Tetrazolium): 50 mg/ml in 0.7 ml DMF and 0.3 ml H2O

BCIP: 25 mg/ml in 1 ml DMF (N,N-Dimethyl-Formamide)

10X TBS: 0.5 M Tris, 1.5 M NaCl, pH 7.5

TTBS: 1X TBS + 0.02% Tween-20

Transfer buffer: 25 mM Tris, 192 mM Glycine, 20% Methanol

For 1 L 3.03g 14.41g 200 ml

Dye buffer: 100 mM Tris, 100 mM NaCl, 5 mM MgCl2, pH 9.5 Or 100 mMNaHCO3, 1 mM MgCl2, pH 9.5

2X sample buffer: 50-200 mM Tris-HCl, pH6.8-7.5

SDS: 2-4%

Glycerol: 15-20%

Bromophenol blue: 0.1 mg/ml

DTT: 200 mM or B-Me: 5% (add fresh)


14/40

AGAROSE GEL ELECTROPHORESIS:

Casting Gel (0.8-1.2%):

Melt agarose thoroughly in 1X TAE running buffer (40-60 ml for small gel; 100-150 for big gel),

cool to 50-60 C, add 2 ul of 10% ethidium bromide, mix well and cast gel.

Running Gel:

Take 10 ul of sample, add 1-2 ul of loading buffer, mix well and load onto the gel. Load 2-3 ul

of ladder (1 KB) in one lane as standard.

Run the gel for about 20-60 min at 100 V. Take picture.


15/40

Loading Buffer (6X):

0.25% bromophenol blue0.25% xylene cyanol FF (some buffer without this dye)

30% glycerol or 40% sucrose

in H2O

Ethidium bromide:

10% ethidium bromide in H2O (stored at RT in dark bottle). Gel can be stained after run

(immerse the gel in 0.5 ug/ml EB for 30-45 min at RT and destain is not needed).

Caution: EB is a powerful mutagen and also moderately toxic. Ware gloves!

Sequencing with BigDye Terminator (V3.1)

1. In 0.2 ml PCR tube, prepare following mix:

BigDye Reaction Premix (at –20 C) 2 ul

BigDye 5X Buffer (at 4 C) 1 ul

Primer (5 pmol/ul) 1 ul

Template (mini or midi) 1 ul


16/40

H2O 5 ul

2. Perform thermal cycles:

96 C for 4 min, then 35 cycles of:

96 C 20 sec50 C 5 sec (this C depends on primer)

60 C 4 min

3.Clean up:

Spin down the reaction, add 10 ul H2O to bring the

volume to 20 ul, then do mormal clean-up as follows or column

clean-up on next page.

a) Transfer to 1.5 ml tube.

b) Add 2 ul 125 mM EDTA, 2 ul 3M NaAc (pH 5.4) and 50 ul

100% EtOH. Mix well and leave for 15 min at RT.

c) Spin 30 min (max rpm, RT).

d) Wash twice with 300 ul 70% EtOH.

e) Dry the sample and send for sequencing.

Note:If the template is PCR product, we need to do gel

purification or go through Centricon to get rid of any leftover

primers.

Northern-Blot


17/40

Purify RNA

Use Rneasy Plant Mini Kit (Qiagen) to purify total RNA. Use 100 mg tissue and the

final elution volume is 50 ul. If you need to do quantitative comparison, you need to measure the

OD260 to make sure equal loading later. Store at -80 C.

Run RNA denaturing gel

1. Clean gel tray, comb and gel tank carefully to get rid of RNase with method A or B:

A, wash with water, spray with Scrubbing Bubbles, sit for a few minutes, rinse with

water, spray with RNase Away, sit for a few minutes, rinse with autoclaved water; B,

soak with warm 0.1% SDS plus 10 mM EDTA (pH 8.0) for more than 1 h, rinse with

autoclaved water. Put a piece of Saran wrap under the gel tray before casting the gel.

2. Prepare 1% agarose gel: MOPS, water and glassware need to be autoclaved.

For big tray For mini tray

Agarose 2 g 0.5 g

10X MOPS buffer 20 ml 5 mlWater 146 ml 36.5 ml

Melt the agarose and let it cool to around 60 C; then add formaldehyde.

Formaldehyde (37%) 34 ml 8.5 ml

Mix well and cast the gel.

Running buffer is 1X MOPS buffer (for mini gel, need about 250 ml).Load the

sample and run the gel.

3. Sample preparation:

For each sample (6 ul), prepare following: 5X 10X 15X 20X

Formamide 12.5 ul 62.5 125 187.5 250

10X MOPS buffer 2.5 ul 12.5 25 37.5 50Formaldehyde (37%) 4 ul 20 40 60 80

EtBr (10 mg/ml) 0.1 ul 0.5 1 1.5 2

Total 19.1ul/sample

Mix with the sample, incubate at 65 C for 5 min, then chill on ice. Add 2.5 ul loading

buffer (50% glycerol containing 0.1 mg/ml bromophenol blue) and load onto the gel.

Treat the RNA ladder the same way as for samples (ladder+EB+LB—65 C-5 min).

4. Run the gel at 100 V until the dye reach the end (~1.5 h). Afterwards, visualize the

RNA under UV light and take a picture. If you load a ladder lane, put the gel over a

UV box and puncture the ladder band. Also trim a corner off under the ladder lane tomark the orientation.

5. Place the gel in a container with 10X SSPE buffer and gently shake for 15 min

(optional). The gel is ready to go to blot.


18/40

Transfer RNA to membrane (refer to the diagram)

1. Wear clean gloves to cut the Hybond-N+ membrane (gel size) and 3MM paper (4

sheets of gel size and a long one for bridge) and soak them in transfer buffer (10X

SSPE, 300ml).

2. Set up a platform in a glass dish and use the long 3MM paper to make a bridge overthe platform. Use a Pasteur pipette to drive any air out (repeat this for the gel,

membrane and 3 MM paper).

3. Put 2 sheets of 3MM paper over the bridge, then lay the gel (bottom up) on top.

Cover the top of the glass dish with Saran wrap, then cut it open along the edges of

the gel with a blade.

4. put the membrane on the gel followed by another 2 sheet of 3MM paper. Place a

stack of paper towels (2-3 inch thick) on top of the 3MM paper.

5. Place a glass plate and a weight (a bottle of 800 ml water) on top. Allow the transfer

to proceed overnight.

6. After transfer, turn around the membrane with the gel. Use a pencil to mark the

ladder bands on the membrane and trim a corner off. Air-dry the membrane, then fix

the RNA by UV crosslink (auto-crosslink). Membrane can be stored at this point for

a few days or do hybridization right away.

3ong "44 paper

Saran wrap

5el

4erane"44 paper

Paper towel

5lass plate

Weight

5lass plate

Support

5lass dish withtransfer uffer


19/40

Probe preparation

Use Rediprime II Random Prime Labelling System (Amersham/GE Healthcare) to

prepare the probe.

1. Use PCR product of your gene of interest or cut out your gene from a plasmid. In

either case, you need to gel purify your product (final elution in 30 ul) and check the

concentration.

2. Follow the Protocol to do the labeling.

Hybridization

1. Put your cross-linked membrane into a hybridization tube (or 50 ml tube for mini-

gel). Add 5 ml (for 50 ml tube) Ultrasensitive hybridization buffer (Ambion, #8670;

warm the buffer up at 65 C first) and pre-hybridize for 1 h at 42 C.

2. Add your labeled probe into the buffer (be sure do not add the probe directly to the

membrane). Hybridize the membrane with the probe overnight at 42 C.

3. Wash the membrane for 5-10 min with 2X SSPE/0.1% SDS at 42 C. Wash one more

time.

4. Wash the membrane for 15 min with 0.1X SSPE/0.1% SDS at 42 C. Wash one more

time.

5. Wrap the membrane in Saran wrap.

6. Expose the membrane to Phosphor screen overnight (or longer if the signal is not

strong).

7. Visualize the image using a PhosphorImager.

Buffers (need to be autoclaved)

1. 20X SSPE buffer: 3 M NaCL, 0.2 M NaH2PO4, 0.02 M EDTA, pH7.4.

2. 10X MOPS buffer: 0.2 M MOPS, 0.1 M sodium acetate, 0.01 M EDTA, pH 7.0.

3. Pre-hybridization and hybridization buffer: Ultrasensitive hybridization buffer from

Ambion (#8670).

4. 20X SSC buffer (replace SSPE with SSC for Southern): 3 M sodium chloride, 0.3 M

sodium citrate, pH 7.0


20/40

Southern-blot (Hybridization analysis of DNA)

Purify total DNA and enzyme digestion

Purify Total DNA from plant.

Digest the DNA into small fragments (use 200ul digestion system, then concentrate to 40 ul).

Run gel

Load the sample on ~7 mm thick 0.7% agarose gel with EB (load DNA + a little loading buffer,

run gel for 30 min at 70 V), then submerge the gel totally and run at ~20 V overnight.

After running the gel (take a final picture), punch the ladder bands on UV box, then cut a corner

under the ladder lane.

Depurination (Optional, since the fragments larger than 15 Kb are hard to transfer)

Wash the gel in 0.2 M HCl for 5 (mini gel) to 10 (large gel) min on a shaker, then rinse with

water.

Denaturation

Wash the gel in denaturation buffer for 30 min on a shaker, then rinse with water. Transfer the

gel into neutralization buffer for 30 min on a shaker. Now the gel is ready to go to blotting.

Blotting and hybridization

Same as in Northern (use SCC instead of SSPE).

Buffers

1. 20X SCC buffer: 3 M sodium chloride, 0.3 M sodium citrate, pH 7.0.

2. Depurination solution: 0.3 M HCl.

3. Denaturation solution: 1.5 M NaCl, 0.5 M NaOH.

4. Neutralization buffer: 1.5 M NaCl, 0.5 M Trizma base, pH 7.5.

Note: 5enoic D6 is digested with one or ore restriction en7yes$ and the resulting fragents areseparated according to si7e y electrophoresis through an agarose gel. The D6 is then denatured intosinglestranded in situ and transferred fro the gel to a erane. The D6 attached to the eraneis hyridi7ed to radiolaeled D6 or #6$ and autoradiography is used to locate the positions of andscopleentary to the proe.


21/40

cDNA synthesis

1. Linearize RNATotal RNA 3 ul

10 mM dNTP 1 ul

3’ Primer (50 pmoles/ul) 0.5 ul

DEPC-treated H2O 3.5 ul

Total 8 ul

Incubate the sample at 65 C for 5 min, then place on ice.

2. Prepare a master mixture5X RT buffer 4 ul

25 mM MgCl2 4 ul

0.1 M DTT 2 ul

RNaseOUT 1 ul

SuperScript II 1 ul

Total 12 ul

3. SynthesisMix master mixture with linearized RNA, then go to

42 C 60 min

72 C 15 min

Chill on ice.

Brief CF. Add 1 ul RNase H, then incubate at 37 C for 20 min.


22/40

Purification of His-tagged protein expressed from insect cells(Under native conditions)

1. Cells were harvested 48-72 h post inoculation (with 5 MOI) by CF (2000 g for 5

min).

2. Flash-freeze the pellet in liquid N, then resuspend the cells in lysis buffer (1 ml for 6or 12 well plate; 5 ml for 25 cm2 flask; 10 ml for 75 cm2 flask).

3. Cells were lysed by three cycles of freezing and thawing (liquid N---37 C).

4. Using a 20-gauge double-lock needle and 2 syringes, shear the sample by passage

through the needle 3-4 times.

5. Cell debris was pelleted by CF at 8,000 g for 10 min (save 50 ul supernatant for

sample 1; part of pellet for sample 2).

6. Add nickel-resin (equilibrated with washing buffer; use 100 ul for 1 ml lysis buffer,

250 ul---5 ml , 500 ul---10 ml) into the cleared lysate.

7. Load the lysate with resin on an empty column with the outlet closed. After the resin

settled down, open the outlet and collect the flow-through. Let the flow-through passthe column one more time (collect 50 ul for sample 3).

8. Wash the resin with washing buffer (3 volume of the lysate, sample 4).

9. Elute the protein with elution buffer (1/5 volume of the lysis buffer, collect 25 ul for

sample 5).

10. Elute once more (collect 25 ul for sample 6).

11. Concentrate the combined elute to about 1 ml with Centricon-30 at 6000 rpm (sample

7).

Note: Try to keep samples at 4 C or on ice to prevent proteolysis.

Cells also can be lysed by adding 0.1% Triton X-100 into lysis buffer. Vortex cells in the

lysis buffer to suspend and break the cells. Incubate the samples on ice for 30-45 min (vortex at

10 min intervals to assist lysis).

Buffers:

Lysis buffer


23/40

50 mM NaH2PO4, 300 mM NaCl, 5 mM imidazole, 0.05% Tween 20, pH

8.0, add protease inhibitor before use (10 ul/ml)

Washing buffer


8.0

Elution buffer

50 mM NaH2PO4, 300 mM NaCl, 200 mM imidazole, 0.05% Tween 20,

pH 8.0

Purification of native protein expressed from insect cells(Under native conditions with DEAE-column)

1. Cells were harvested 48-72 h post inoculation (with 5 MOI) by CF (2000 g for 5 min).

2. Flash-freeze the pellet in liquid N, then resuspend the cells in lysis buffer (1 ml for 6 or

12 well plate; 4 ml for 25 cm2 flask; 10 ml for 75 cm2 flask; 20 ml for 225 cm2 flask).

3. Cells were lysed by three cycles of freeze (liquid N2) an d thaw (37 C).



5. Cell debris was pelleted by CF at 8,000 g for 10 min (save 20 ul supernatant for sample

1; part of pellet for sample 2).

6. 10 ml of the cleared lysate was load onto a DEAE Sepharose Fast Flow column at a flow

rate of 0.5 ml/min. After loading the sample, run for 10 min with carrying buffer (20 mM

Tris, pH 8.0 plus 50 mM NaCl) only without collecting.

7. Elute the protein with Tris,-NaCl solution (20 ml of each of following NaCl: 200 mM---

250 mM---300mM---350 mM--- 400 mM---500 mM) at a flow rate of 0.5 ml/min.

Collect a fraction for every 10 min (4 fractions/NaOH concentration). Take 6 ul to

measure OD260 and take 25 ul sample to prepare for Western (add 25 ul of 2X PAGE

sample buffer, boil for 10 min, load 10 ul/lane).

8. Combine the fractions with the protein and load onto Centricon-30. CF at 6000 g at 4 C

to the final volume of 1 ml.


Cells also can be lysed by adding 0.1% Triton X-100 into lysis buffer. Vortex cells in the

lysis buffer to suspend and break the cells. Incubate the samples on ice for 30-45 min (vortex at

10 min intervals to assist lysis).


24/40

Buffers:

Lysis buffer

20 mM Tris, 20 mM NaCl, pH 8.0, add protease inhibitor Cocktail before

use (5 ul/ml, HaltTM Protease Inhibitor Cocktail EDTA-Free, Pierce)

Elution buffer20 mM Tris, pH 8.0 plus different concentration of NaCl. Prepare 25 ml

of each and use ~20 ml.

Reference:

1. Mavrakis et al. 2003. Isolation and characterization of the Rabies virus No-P

complex produced in insect cells. Virology 305:406-414.

2. Gupta et al. 2003. Identification of a novel tripartite complex involved in replication

of Vesicular stomatitis genome RNA. J. of Virol. 77: 732-738.

Purification of His-tagged protein expressed from bacteria(Under native conditions)

1. Transfer a single colony (be sure from an expression strain) to a culture tube containing 4

ml of medium with antibiotics, and grow o/n at 37 C on a shaker.

2. Inoculate 2 flasks of medium (with antibiotics) with the o/n culture (add 2 ml of culture

into 30 ml of medium). Grow the bacteria for 50-60 min (OD = 0.5-0.7) at 37 C with

vigorous shaking (~2h). Grow another flask all the way and pellet the cells as un-induced

control.

3. Add IPTG to a final concentration of 1 mM into the culture and grow the culture for

another 4 h. Take 1 ml culture and pellet the cells to check the protein expression and

solubility, and pellet the rest and store at –20 C for purification.

4. Resuspend the cells in 10 ml lysis buffer and sit on ice for 20 min. Cells were lysed by

sonication (3 X 20 sec at high setting) on, then using a 20-gauge double-lock needle and

2 syringes, shear the sample by passage through the needle 3-4 times.



6. Add nickel-resin (equilibrated with washing buffer; use 100 ul for 1 ml lysis buffer, 500

ul---10 ml , 750 ul---15 ml) into the cleared lysate and mix gently for 30 min on a shaker.

7. CF 2 min at 4000 rpm and discard the supernatant (sample 3---unbound).

8. Wash the resin twice with 10 ml of washing buffer by CF.

9. Load the lysate with resin on an empty column with the outlet closed. After the resinsettled down, open the outlet and drain the flow-through (collect 50 ul for sample 4---

wash).

10. Elute the protein with elution buffer (1/5 volume of the lysis buffer, collect 25 ul for

sample 5).


12. Concentrate the combined elute to about 1 ml with Centricon-30 at 6000 rpm (sample 7).


25/40


Lysis buffer

50 mM NaH2PO4, 300 mM NaCl, 0.05% Tween 20, 0.1% Triton X-100,

pH 8.0, plus 100 ug/ml lysozyme (made fresh)

Washing buffer


8.0

Elution buffer

50 mM NaH2PO4, 300 mM NaCl, 200 mM imidazole, 0.05% Tween 20,

pH 8.0

Purification of His-tagged protein expressed from bacteria(Under native conditions with BugBuster-from Novagen)

1. Resuspend the cells (from 40 ml culture) in 5 ml of BugBuster.



3. Cell debris was pelleted by CF at 8,000 g for 10 min (save 50 ul supernatant for

sample 1; part of pellet for sample 2).4. Add 1 ml of nickel-resin (equilibrated with washing buffer) into the cleared lysate

and mix gently for 30 min on a shaker.




settled down, open the outlet and drain the flow-through (collect 50 ul for sample 4---

wash).

8. Elute the protein with 2 ml of elution buffer (collect 25 ul for sample 5).


10. Concentrate the combined elute to about 1 ml with Centricon-30 at 6000 rpm (sample7).



26/40

Washing buffer


8.0

Elution buffer50 mM NaH2PO4, 300 mM NaCl, 200 mM imidazole, 0.05% Tween 20,

pH 8.0

Purification of His-tagged protein expressed from bacteria(Under denatured conditions)

1. Transfer a single colony (be sure from an expression strain) to a culture tube containing 4

ml of medium with antibiotics, and grow o/n at 37 C on a shaker.

2. Inoculate 2 flasks of medium (with antibiotics) with the o/n culture (add 2 ml of culture

into 40 ml of medium). Grow the bacteria for 50-60 min (OD = 0.5-0.7) at 37 C with

vigorous shaking. Grow another flask all the way and pellet the cells as un-induced

control.

3. Add IPTG to a final concentration of 1 mM into the culture and grow the culture for

another 4 h. Take 1 ml culture and pellet the cells to check the protein expression, and

pellet the rest and store at –20 C for purification.

4. Resuspend the cells in 10 ml lysis buffer. Cells were lysed by sonication (3 X 30 sec at

high setting and take care to avoid frothing). The lysate should be translucent when lysisis complete. Using a 20-gauge double-lock needle and 2 syringes, shear the sample by

passage through the needle 3-4 times.



6. Add 500 ul of nickel-resin (equilibrated with washing buffer) into the cleared lysate and

mix gently for 30 min on a shaker.


27/40




settled down, open the outlet and drain the flow-through (collect 50 ul for sample 4---

wash).

10. Elute the protein with elution buffer (1/5 volume of the lysis buffer, collect 50 ul forsample 5).


12. Add 2 ml H2O to combined elute and concentrate it to about 1 ml with Centricon-30 at

6000 rpm at 10 C (take 50 ul for sample 7).

Buffers:

Lysis buffer

100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea, pH 8.0

Washing buffer100 mM NaH2PO4, 10 mM Tris-Cl, 6 M urea, pH 6.3

Elution buffer

100 mM NaH2PO4, 10 mM Tris-Cl, 6 M urea, pH 4.5

Note: Due to the dissociation of urea, the pH of the above buffers should be adjusted

prior each purification. Do not autoclave.

Check the protein expression from bacteria

1. Transfer a single colony (be sure from an expression strain) to a culture tube containing 2 ml of

medium with antibiotics, and grow o/n at 37 C on a shaker.

2. Inoculate 2 tubes containing 4 ml of medium with the o/n culture (200 ul of culture/tube). Grow

the bacteria for 40-50 min (take 1 ml to check OD = 0.5-0.7) at 37 C with vigorous shaking.

3. Add IPTG to a final concentration of 1 mM into one tube and grow the culture for another 3 h.

Leave the other tube as uninduced control on the shaker.

4. Harvest cells from 1.0 ml culture by CF, then resuspend the pellet in 200 ul of SDS-PAGE

sample buffer. Boil the sample for 10 min, then CF for 10 min and load 10 ul of the supernatantonto the gel. Use uninduced as comparison.

5. After Coomassie staining, compare the band pattern for protein expression (total protein).

6. Harvest cells from another 1.0 ml culture by CF, then freeze the cell pellet for checking the

protein solubility.


28/40

Note:

A, We transform normal (ex. Top 10) cells with our construct first. After confirming the

right construct, it needs to be transformed into an expression E. coli strain (such as BL21

or Rosetta).

B, If use Rosetta cells, LB should contain 35 ug/ml of chloramphemicol plus anotherantibiotics which your plasmid resistant to.

C, Solubility tests: Resuspend the frozen cell pellet in 200 ul of BugBuster (Novagen).

Incubate for 10 min at RT with shaking, then CF for 5 min. Supernatant should be

soluble and pellet should be insoluble. Add SDS-PAGE sample buffer (1:1 to

supernatant and 100 ul to pellet) and load 10 ul/lane to run the gel.

Purification of P by passing through Sephacryl column

Column length: 27 cm (after packing)

Column diameter: 1 cm (inside)

Elution speed: 0.4 ml/min (pump setting: 04)

Sample amount: 1 ml (2.5 min at 0.4 ml/min)

Fraction: 5min/fraction (start collecting after loading the sample)

Elution buffer: 20 mM Tris, pH 8.0

Protein-Blotting Protein-Overlay (Far-Western):

Run SDS-PAGE

Transfer protein onto membrane

Protein-overlay and detection

1. After transfer, membrane was washed with H2O (20 min on shaker at RT) to removeSDS.

2. The membrane was then incubated overnight at 4 C on a shaker with TBS containing 5%

skimmed dry milk to renaturing transferred proteins and blocking nonspecific binding

sites on the membrane. All other incubation and washing were done at RT on a shaker

and incubation buffer (except substrate buffer) contains 5% milk.


29/40

3. The membrane was cut into strips and incubated with cell lysate (1/4 in TBS) expressing

bait protein for 3 h.

4. Wash 3 X 10 min in H2O.

5. Incubate the membrane with primary antibody against bait protein in TBS for 2 h.


7. Incubate the membrane with secondary Ab (make sure use right Ab at proper dilution) inTBS for 2 h.


9. Develop in NBT-BCIP dye solution (cover from light for about 10 min).

10. Rinse with water and air dry the membrane.

Cell lysis:

1. For insect cell: Resuspend the cells (T75 flask) in 15 ml of lysis buffer (20 mM Tris,

20 mM NaCl, pH 8.0 containing 0.1% Triton X-100). Using a 20-gauge double-lock

needle and 2 syringes, shear the sample by passage through the needle 3-4 times.

Add 15 ml of glycerol, mix well and store at –20 C.

2. For E. coli cell: Resuspend the cells (40 ml culture) in15 ml of TBS plus 0.1% Triton-

X100). Lyse the cells by sonication (3 X 30 sec). Add 15 ml of glycerol, mix well

and store at –20 C.

Phosphoprotein Gel Staining

1. Run two identical gels (normal SDS-PAGE).

2. Fix the gel in 50 ml fixer overnight.

3. Wash the gel in H2O (10 min X 3 times). Afterwards, one gel for phosphoproteinstaining (Pro-Q Diamond Phosphoprotein Gel Staining), and the other for total protein

staining (SYPRO Ruby Protein Gel Staining).

Phosphoprotein Gel Staining

1. Stain the gel in the dark in 50 ml of Pro-Q staining solution for 75 min.

2. Destain the gel in the dark in 90 ml of Destain solution (30 min X 3).


30/40

3. Wash the gel with H2O (5 min X 2).

4. View the bands (Set bottom box ON and SYBR Gold CY3 or EB).

5. Gel bands fade after 2-3 h.

Total Protein Gel Staining

a. Stain the gel in the dark in 50 ml of SYPRO Ruby staining solution for 5 h orlonger.

b. Destain the gel in the dark in 90 ml of Destain solution (30 min X 2).

c. Transfer the gel into H2O (bands do not fade for days).

d. View the bands (Set bottom box ON and EB or SYBR Gold CY3).

Note: Perform all fixation, staining and washing steps on an orbital shaker at 50 rpm in a large

plastic weighing dish.

Solutions:

Fixer50% methanol

10% acetic acid

Destain (for total protein)

10% ethanol or methanol

7% acetic acid

Leader binding gel shift assay

PCR:

Design a pair of primers (5’ with T7 promoter plus a cloning site Xba I; 3’ with a blunt-end

enzyme site SnaBI plus a cloning site Hind III).

Clone into pUC19 (no promoter):

Clone into pUC19 at Xba I and Hind III. Do mini + midi prep.


31/40

Linearize the template:

plasmid-DNA (2-5 ug) 20 ul

buffer 4 10 ul

enzyme 2 ulH2O 68 ul

Total 100 ul

Incubate for 3 h at 37 C.

Do phenol-Chloroform extraction; ethanol precipitation, and

resuspend in 20 ul of H2O then use 10 ul to do transcription.

RNA transcription:

Epicentre kit

Keep all components on ice and combine the reaction mixture at RT in the following order.Cold: Hot

H2O 2.8 ul 0.8 ul

Linearized template (1 ug) 10 ul 10 ul

10X AmpliScribe T7/T3 Reaction Buffer 2 ul 2 ul

100 mM ATP 0.75 ul 0.5 ul

100 mM CTP 0.75 ul 0.5 ul

100 mM GTP 0.75 ul 0.5 ul

100 mM UTP 0.75 ul 1 mM 0.5 ul32P-UTP --- 2 ul

RNase inhibitor (RNAguard) 0.2 ul 0.2 ul

100 mM DTT 2 ul 2 ul

AmpliScribe T7 enzyme 1 ul 1 ul

total 20 ul

Incubate for 2 h at 37 C, then clean up (phenol extraction…..). For “cold” test, take 5 ul

(before clean up) and run gel (150 V---15 min).

Clean-up:

1. Add 90 ul H2O, 50 ul H2O-saturated phenol, 50 ul chloroform.

2. Vortex thoroughly, then CF for 5 min.

3. Pipette 95 ul aqueous phase (top) into a new tube

4. Add 300 ul Isopropanol:10 M NH4Ac (10:1) solution and mix well.

5. Incubate for 30 min or longer at –20 C.

6. CF for 15 min at max at 4 C.


32/40

7. Pour off solution, and add 700 ul 70% ethanol

8. CF for 10 min at max at 4 C.

9. Pour off ethanol thoroughly

10. Air dry the pellet for 15-20 min

11. Dissolve the pellet in 200 ul of 1X TE add 0.5 ul RNase inhibitor. Aliquot to 40

ul and store at –20 C.Gel shift assay:

Binding reaction:

Binding Buffer 4.8 ul

RNAguard 0.2 ul

tRNA (0.1 mg/ml) 1 ul

Protein (in glycerol) 2 ul32P-labeled RNA 2 ul

Incubate for 15 min at RT. Add 2 ul loading buffer, load all onto the gel and run non-

denaturing PAGE with 1X TAE for 1 h at 4 C. After the run, transfer the gel onto 3 M filter

paper, cover with a plastic sheet and vacuum dry it.Check the radioactivity with a Geiger counter and exposed to the cleaned Phosphor

screen for 3 h-o/n.

5% PAGE non-denaturing gel (10 ml for 2 small gels):

H20 8.00 ml

50X TAE buffer 200 ul

30% acrylamide/bis (37.5:1) 1.67 ml

10% APS 100 ul

TEMED 8 ul

Binding buffer: 200 mM Na-PO4, pH 7.5 containing 600 mM NaCl.

dsRNA transcription (follow Stratagene’s Dicer enzyme):

A. Design PCR primers with T7 promoter in both primers and run PCR to produce the

transcription template.

5el dryer ed

2 sheets of paper

"44 paper

gel

Saran wrap


33/40

B. Check the PCR concentration and use 1 ug to do transcription.

C. Use Epicentre kit to do transcription (make up to 20 ul with H2O).

D. Clean-up as RNA transcription.

DNA binding assay:A. Design PCR primers without T7 promoter in both primers and run ‘cold’ PCR.

B. Check the ‘cold’ PCR product by running the gel.

C. Run ‘hot’ PCR (1 ul of 10 mM dATP, dGTP, dTTP and 0.1 mM dCTP plus 5 ul P32-

dCTP).

D. Clean-up as DNA (add 50 ul H2O, 50 ul phenol, 50 ul chloroform, CF 5’ then do EtOH

precipitation and resuspend in 200 ul H2O).

Northwestern blot (Zehner et al., 1997. Nucleic Acids Res. 25: 3362- )

Run SDS-PAGE

Transfer to nitrocellulose membrane

Binding assay1. After transfer, membrane was washed with H2O (2 X 10 min on shaker at RT) to remove

SDS.

2. The membrane was then incubated overnight at 4 C on a shaker with TBS containing 5%

skimmed dry milk to renaturing transferred proteins and blocking nonspecific binding

sites on the membrane. All other incubation and washing were done at RT.


4. Incubate the membrane with 32P-labeled RNA probe against bait protein in binding buffer

for 2 h.

5. Wash 3 X 10 min in binding buffer.

6. Dry the membrane, check the radioactivity with a Geiger counter and exposed to the

cleaned Phosphor screen for 3 h-o/n.

Binding buffer:

50 mM Tris-HCl pH7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 5 U/ml

RNasin


34/40

NICOTIANA BENTHAMIANA CALLUS CULTURE

Note: Work under aseptic conditions! Sterilize all solutions and accessories prior to using

them!

A. Germination of N. benthamiana seeds under aseptic conditions

Wash down hood thoroughly with 10% bleach solution before starting!

Surface sterilize Nicotiana benthamiana seeds:

1.Fill up two sterile graduated 1.5 ml eppendorf tube to ~ 50µl line with N.

benthamiana seeds.

2.Add 700 µl 70% ethanol, close tube and shake it several times. Wait a few seconds

for seeds to settle down on the bottom of the tube. Remove (discard) ethanol with

a sterile 1 ml pipet tip (use new tip for each tube)

3.Repeat the above step 4 more times.

4.Add700 µl undiluted Clorox bleach and35 l 10% SDS. Shake tube up and down

for 9-10 min. Wait for seeds to settle down. Remove bleach solution (use new tip

for each tube).

5.Add 700 µl sterile dH2O. Invert tube a few times, wait for seeds to settle down, then

remove dH2O. Every time use a fresh tip for each tube. Repeat this step 4 more

times (5 rinses total). Finally, add350 l dH2O to seeds in the tube. Use a sterile

large orifice 1 ml tip to place seeds on two MS-E plates (1 plate for each tube).

Spread/separate seeds as much as possible using sterile bacterial loops. Do not dry

plates after spreading seeds.

Seal plates with parafilm. Incubate plates at 23 Co, 16 h light and 8 h dark.

B. Induction of N. benthamiana callus culture

UseMS-E plates for N. benthamiana. Use 100X20mm petri plates. Can store plates for

several weeks at 4C.


35/40

First passage can be made 3-5 weeks after plating. Cut leaves with a sterile scissors,

induce wounds by squeezing leaves w/sterile forceps and pressing gently onto medium

(MS-E).

Seal plates with parafilm. Incubate plates at 23 Co

, 16 h light and 8 h dark.

C. Maintainance of N. benthamiana callus culture on MS

UseMS plates for N. benthamiana callus maintenance. Let the plates dry under the

hood with the lid partially covering a dish. Longer storage at 4C is okay.

From this point on onlyMS medium is used.

Callus should be light yellow with a little bit of green. Brown color indicates dead or

dying tissue. Good callus is friable but not too soft. Transfer only healthy looking callus!

Do not passage callus which is too soft or brownish. 1 plate will make 2-3 new plates by

dividing callus into ~ 3 mm pieces with forceps. Transfer ~ 20 such pieces into a fresh

plate. Distribute pieces evenly on the plate.

Germination: 3 weeks (seeds plated onMS-E, 2 plates)

1st passage: 4 weeks (transfer seedlings onMS-E toMS,usually 5-6 plates) =1P

2nd passage: 3 weeks =2P (usually 5-6 plates)

3rd passage: 2 weeks =3P

4th passage: 2 weeks =4P

5th passage: 2 weeks =5P

Seal plates with parafilm. Incubate plates at 23 Co, 16 h light and 8 h dark.

The 3rd, & 4th and 5th(though 5th seems to be less valuable) ) passages seem to be

best for protoplast isolation (Zivile).


36/40

Primer design

Size: 22-25 bp (or longer). Size will determine the melting temperature (Tm).

Tm = 2 x (A+T) + 4 x (C+G); Normally Tm should be higher than 55 C.

If the primer is too short, Tm will be very low, and the specificity will be reduced.

When calculating Tm, do not count the modified bps plus 2-3 bps of 5’ protecting ends.

Sequence: The 3’ end of the primer is the most important part for the amplification. It must be

strictly complementary to the template sequence, and if possible, have a high G+C content.

Sequence modification (restriction site, mutation site, site-directed mutagenesis): Always keep a

complementary 3’ end and try to introduce the modification site at the 5’ end (leave 2-3complementary bp to protect the site). For site-directed mutagenesis, introduce the modification

site in the middle of both primers.

Special rules:

A, If you are given a sequence (single strand DNA), this sequence is (+) sequence.

The 3’ primer should be complementary to the sequence; and the 5’ primer should be the

same sequence as the template (the given sequence).

For example: Given sequence

5’-CATGCCTTAAGCCCG..................................... TTCCATGGAACTGTT-3’ 5’-CTTAAGCCCG-3’ 3’-GGTACCTTGACAA-5’

5’ primer 3’ primer

B, A given virus sequence (single strand DNA) is the same sequence as mRNA (with

U replaced by T). For example: To design a 5’ primer with a start codon (5’AUG3’) and

a 3’ primer with a stop codon (5’UGA3’):


37/40

Given sequence

5’-CATGC CTTAAGATACGTC....................CGTCATTCCATGGACTT GTG-3’

5’-CA ATG CTTAAGATACGTC-3’...........3’-GCAGTAAGGTACCT ACT CAC-5’


C, If you need to add enzymes to your primer, follow enzyme 5’ to 3’. For example:

To design a 5’ primer with BamH I (5’GGATCC3’) and a 3’ primer with AvrII

(5’CCTAGG3’):

Given sequence

5’-GCTTAAGATACGTC.............CGTCATTCCATGGGTG-3’

5’-GGATCCGCTTAAGATACGTC-3’....3’-GCAGTAAGGTACCACGGATCC-5’


Our lab use 0.5 ul of 50 pmoles/ul to do PCR. If problem happened and primers are suspected,

load 5 ul of 50 pmoles/ul onto gel and run for 10 min at 120V to check.

ds RNA transcription and confirmation (follow Dicer Enzyme Manual from Stratagene)

To confirm ds RNA:

2 ul ds RNA transcription product

1.5 ul 2 M NaCl (final 0.3 M)

or 1.5 ul 0.2 M NaCl (final 0.03 M)

1 ul 0.5 mg/ml RNase A

5.5 ul TE

37 C for 1 h, then run the gel. ds RNA will be digested by RNase A under low salt

condition, but not high salt condition.


38/40

Purification of His-tagged protein expressed from plant(Under native conditions)

1. Leaves were harvested 3-4 days post infiltration.

2. Leaves were ground with a chilled mortar and pestle in extraction buffer

(leaf:buffer = 1:5).

3. CF the homogenate for 15 min at 10,000 rpm at 4C. Filter through Miracloth

(take 50 ul supernatant and add into 25 ul sample buffer as sample 1.)

4. Add 250 ul nickel-resin (equilibrated with extraction buffer) into the supernatant

(6 ml His-tag sample + 6 ml none-tag sample).

5. Mix for 30 min-1 h on a shaker on ice.

6. CF 2 min at 4000 rpm and discard the supernatant.

7. Wash the resin twice with 10 ml of washing buffer by CF. Transfer resin with 1

ml washing buffer into an eppendorf, spin and remove the supernatant.

8. Add 500 elution buffer into resin and incubate for 10 min.

9. CF and collect supernatant (50 ul + 50 ul SB as sample 2).

10. Take 50 ul resin add to 50 ul SB as sample 3.

Extraction buffer

300 mM NaH2PO4, 0.1% B-ME, pH 7.5


39/40

Washing buffer

100 mM NaH2PO4, 100 mM NaCl, 5 mM imidazole, pH 7.5

Elution buffer

50 mM NaH2PO4, 100 mM NaCl, 200 mM imidazole, pH 7.5

RNA Purification with RNAzol RT (Molecular Research Center)(All CF is done at max speed at Rm T)

1. Grind 70-100 mg leaf in liquid nitrogen with a mortar and pestle.

2. Add 1 ml of RNAzol RT reagent (Cat #: RN 190) and grind more. Add 400 ul

H2O, mix well, transfer into a 1.5 ml tube and sit for 15 min at Rm T.

3. CF the homogenate for 15 min.

4 Transfer 1.1 ml of the supernatant to a fresh tube (go to 5a or 5b from here).

5a Add 440 ul of 75% ethanol, mix well and sit for 5 min.

6a CF 10 min. (Save 800 ul supernatant for small RNA isolation)

7a Wash the pellet twice with 400 ul of 75% ethanol (CF for 2 min each time).

8a Remove all left-over ethanol and dry the pellet at Rm T for 15 min.

9a Dissolve the RNA in 50 ul of H2O (repeated pipetting).

5b Add 60 ul chloroform to the supernatant (from step 4), vertex for 15 sec and CF

10 min.

6b Transfer 800 ul of aqueous phase (no color) into a new tube (if you want total

RNA, go to 5c)

7b Add 320 ul of 75% ethanol, mix well and sit for 5 min.


40/40

8b Do the same as 6a through 9a

5c Add 640 ul of isopropanol to the aqueous phase (from step 6b), mix well and sit

for 10 min.

6c CF 10 min.7c Do the same as 7a through 9a

Small RNA isolation

1. Add 640 ul of isopropanol to 800 ul supernatant saved for 6a, mix well and store for 30

min at 4 C.2. CF 15 min.

3. Wash the pellet twice with 400 ul of 70% isopropanol.

4. Remove all left-over isopropanol and dry the pellet at Rm T for 15 min.

5. Dissolve the RNA in 50 ul of H2O (repeated pipetting).

MG Lab Protocols

Documents

Transcript of MG Lab Protocols