(1) Soil preparation protocol     

(edit by MMQ 4/24/12)

1.Autoclave soil in autoclavable bags, and then dump the autoclaved soil into the autoclaved tank;

2.Fill the tray with autoclaved soil to 2/3 full;

3.Scoop out some fertilizer (to the black marked line);

4.Dump the fertilizer to a watering bucket, and then add water to 1/4 full, shaking to dissolve the fertilizer;

5.Irrigate the soil of the tray with the water containing the fertilizer (try to do it evenly to well spread the fertilizer into the soil);

6.Mix the soil with scoop, try to shatter clods, add water during this process to soak the soil;

7.Fully fill the flowerpot with the water soaked soil, drain the water away for a little while, and then the flowerpot will be ready to be used.

(2)Protocol for plant transfer from petri dish to pot soil       

(edit by MMQ 4/24/12)

1.Check the plants in the petri dish to see if they reach to 4 to 6 leaves stage that is ready to be transferred into soil;

2.Dig a hole in the pot soil;

3.Use forceps to gently separate one plant from the petri dish (hold the forceps loosely to avoid mechanical damage to the young seedlings);

4.Insert the root part of the plant to the hole and cover it with soil, press the soil a little bit to make the plant stable in the soil;

5.Transfer 4 to 5 plants for each flowerpot;

6.Once one tray is filled, put the tray under the light in the shelf and cover the tray with plastic cover, make sure the two clearstory-like windows in the cover are fully closed;

7.Open the two clearstory window three days after plant transfer;

8. 8.Remove the plastic cover in another three days.
   

9. 9.Fill the tray with water once a week if the pot soil feels light and dry (0.5cm height of water in side the tray is enough);
   

10. 10.Water the plant tray every three days after starting flower.
    

(3) 1/2 x Murashige & Skoog (MS) Medium (1L)

1. To a 1 L bottle, add 2.165 g MS salts with macro- and micro-nutrients (There are MS with phosphate and without phosphate, you need to choose the right one, stored at 4C);

2. Add 10 g sucrose;

3. 3.Add 7-9 g Phytagel (phytablend);
   

4. Add 1 ml of 1000*Vitamin (Vitamin stock should be shaking for a while until crystals disappear);

5. Add ddH2O to 1L and adjust the pH to 6.8;

6. Autoclave at 121 C for 30 min

7. Start the cabinet hood for at least 30 min before pouring plates inside the hood, while autoclaving;

8. Pour the plates inside the cabinet hood, about 30 ml/plate.

9. To make selective plates using proper antibiotics, let medium cool down to about 60 C then add antibiotics; mix well before pour plates;

(Note: some people also use 2 or 3% of sucrose. If you are not going to work on carbon metabolism or certain phytohormones, this is not very important.)

(4) EMS Mutagenesis for Arabidopsis Seeds

1.100 mM phosphate buffer preparation:

1.)Prepare 100ml of 1M K2HPO4 (melt 17.42g Dipotassium phosphate into ddwater, add ddwater to 100ml final volume);

2.)Prepare 100ml of 1M KH2PO4 (melt 13.61g monopotassium phosphate into ddwater, add ddwater to 100ml final volume);

3.)Pour 70ml of 1M K2HPO4 into a beaker;

4.)Add 20ml of 1M KH2PO4 and mix the solution;

5.)Adjust the pH of the mixture to 7.5 (in this step, you will probably need to add 2~3ml of 1M KH2PO4); Then this mixture will be 1M phosphate buffer with pH 7.5.

2.Seed EMS Mutagenesis (small amount):

1.)Put about 0.3ml seeds to the 15ml tube;

2.)Add 9ml of ddwater to the tube and then add 1ml of 1M phosphate buffer;

3.)Screw the cap tightly, mix well and put in 4°C overnight;

4.)Try to remove all the 100mM phosphate buffer;

5.)Add 10ml new 100mM phosphate buffer;

6.)Add 40µl (to a final concentration of 0.4%) of EMS (before adding mix the EMS well);

7.)Screw the cap tightly and airtight it with parafilm;

8.)Incubate the tube in a gentle shaker for 8 hours;

9.)6 hours later, prepare the 1/7 phytablend solution (melt 0.1g of phytablend in 45 ml ddwater in a beaker, boiling in microwave oven, and then pour the  solution to a 50ml tube);

10.)7 hours later, prepare the soils;

11.)8 hours later, wash the seed 20 times with ddwater (10ml/time);

3.Planting Seeds:

1.)Add the washed seeds to the prepared phytablend solution, blow and suck to mix well;

2.)Spread the phytablend solution with seeds drop by drop into the prepared soil;

3.)Cover the tray containing soil pots with cap (keep the clearstories closed);

4.)Keep the tray in cold room for 2~4 days for seed stratification;

4.(To be continued)

5. (5)Flower Dipping
   

1.Agrobacteria culture

1.)Make antibiotics stocks:

a.Kanamycin (stock concentration: 25mg/ml): dissolve 0.25g Kanamycin into 10ml ddwater. Votex to mix well. Filter through a 0.2 micron filter unit and aliquot into 10 1.5ml EP tubes. [labeled as Kana (K), stored in -20oC]

b.Gentamycin (stock concentration: 35mg/ml): dissolve 0.35g Gentamicin into 10ml ddwater. Votex to mix well. Filter through a 0.2 micron filter unit and aliquot into 10 1.5ml EP tubes. [labeled as Gen (G), stored in -20oC]

2.)Make LB+K+G and LB+AGAR+K+G media:

a.LB+K+G: For one liter media weigh 25g LB, melt in ddwater, and make the final volume to 1L. Stir to mix and autoclave for 1hr. After autoclaving, cool at room temperature and stir until the media reach to 55oC, add and mix 1ml of K stock and 1ml of G stock.

b.LB+AGAR+K+G: For one liter media weigh 25g LB and 15g AGAR, melt in ddwater, and make the final volume to 1L. Stir to mix and autoclave for 1hr. After autoclaving, cool at room temperature and stir until the media reach to 55oC, add 1ml of K stock and 1ml of G stock. And then quickly pour the plates.

3.)Grow bacteria:

In cabinet hood, streak out the bacteria from stock or from previous plate; culture the newly streaked out plate in 28oC incubator for two days. (The plates should be LB+AGAR+K+G plates). Then, choose one of the followed two ways:

*At least one day before flower dipping, culture the bacteria in LB+K+G media in 28oC shaker   to shake for about 18 hrs (make sure the OD of the bacteria media is no less than 1).

*preferred: two days before flower dipping, grow a full plate of Agrobacteria.

2.Prepare flower dipping mixture:

1.)Flower dipping inoculation medium preparation: in a sterilized bottle, add 5g sucrose (5% of final concentration), and add ddwater to a final volume of 100ml. Stir to mix well. Autoclave for a long period storage.

2.)Harvest the Agrobacteria: (continue the chosen way)

*centrifuge the LB+K+G bacteria media for 20 mins at room temperature at 5500rpm. Pour out the supernatant, and use pipette to remove the left LB media as much as possible.

*preferred: use knife to scratch the full plate of bacteria and transfer them to a new tube.

3.)Mixture: add 10ml of the flower dipping inoculation medium (5% sucrose solution) into the bacteria tube. Re-suspend and mix the bacteria and medium well. And then add 5 µl of Silwet L-77 detergent, and gently mix.

3.Flower dipping:

Add 50 µl of the flower dipping mixture to every flower bud (be careful not to drip the flower dipping mixture to soil). After flower dipping, lie down the flower pots at 40o in a black box, add about 0.5 cm height of water to the black box, cover the lid well and let them stay overnight. And in the next day, return the pots to the normal shelf and culture under light. If the plants grow well, and if there are new flowers opening, repeat the flower dipping one week later.

6. (6)DNA Sequencing at MTU (Taught by Surendar Dhadi and Summarized by MMQ)
   

1. PCR setup (Sequencing reaction):

x uL water

4 uL sequencing buffer

0.5 uL sequencing primer

100 ng plasmid (if total is 4 kB) or 200 ng plasmid (if total is 8 kb)

0.5 uLBig Dye

--------

20 uL

Note: Plasmid must be very pure: 260/230 must be above 1.3

                260/280 must be above 1.8

99 cycles of 95 oC /15 sec, 46 oC /15 sec, and 72 oC/ 3 min

Store at -20oC for 6 months if desired

DNA template quantity for SEQ PCR:

BP                          ng

100~200                 1~3ng

200~500                 3~10ng

500~1000               5~20ng

1000~2000             10~40ng

≥2000                     40~100ng

2. Sequencing reaction purification:

- Reuse Qiagen gel extraction columns stored in 1M HCl one day before use to destroy DNA (or soak the new column in 1M HCI one day before use), and then put them in sterilized water;

                                                5M HCl: 15 mL concentrated + 85 mL water

1M HCL: Dilute 5 M 1:5 (autoclave before use)

-When using them, spin out HCl, wash 2 x with 500 uL water spin at max speed for 1min for each time of washing; ready to load with Sephadex G-50/25 beads;

-Sephadex G50/25 beads: 50% dilution and autoclave, storing in 4°C. Shake to mix                                                                                                                  before use;

- While stirring Sephadex suspension with a magnet, add Sephadex G-50 suspension to the columns, let stand a few seconds to let beads sink, remove water from top (put it back into Sephadex bottle, add more suspension until the columns are almost full with beads);

- Spin the columns for 3 min at 0.9 g;

- Add one more time Sephadex 50/25 suspension;

- Spin the columns 3 times for 1 min each time at 0.9 g to remove all the water (Orient the columns in the same direction into the centrifuge to not destroy the slant of the Sephadex structure);

- Load the PCR sequencing reaction in the middle of the Sephadex surface

(Usually 18 uL are left);

- Place the column into a 1.5 mL fresh tube;

- Spin 3 min at 0.9 g;

- Now about 13 uL very pure PCR products are through the column into the 1.5 mL   fresh tube;

- Add 7 uL water to regain a volume of 20 uL.

3. Loading the Sequencer:

- Use 0.5 mL tubes, put 20 uL clean sequencing reaction in them, and cut the lids off.

- Up to 8 reactions can be run without rubber lids;

- If more reactions shall be run, rubber lids have to be put onto the tubes;

- Open both doors of the machine;

- Check if all buffers and other liquids are filled enough to sequence your amount of samples, check with someone in Rama’s lab);

- A capillary lasts for 300 runs;

- Use only Pop4 polymer, never Pop6;

- 500 uL Pop4 are loaded in glass tube, gives 50 – 60 sequences;

- Strictly avoid air bubbles in the capillary;

- 2 buffer reservoirs exist, buffer load good for 30 – 40 sequences or 2 – 3 days,   then change buffer;

- Push TRAY button -> Tray moves;

- Add sequence reactions in a row to the tray, starting A1, the A3, then A5, …

- Push TRAY button again, reactions go inside;

- Close both doors.

4. Program the sequencer using the computer:

- Open “310 Data Collection” software;

- File>New>Create New, choose “Regular Sequencing” “Sequence Sample Sheet 48 Tubes”;

- Enter sample name (Start with A1, then A3, then A5, …);

- Choose “Matrix file”:  <none>

- File>Save: Give file name with a date;

- File>New, choose Sequence Injection List (double-click), Sample Sheet (retrieve the    file you just saved from a huge list);

- Pull down proper module: P4StDSeq (1ml) E.mol4;

- Fill down module for all sequences: Mark all (click on the header), then Ctrl-D;

- Run time (min) leave at 32 min if 500 bp, or more set to 40 min if 1 kb;

- Injection seconds: Go up to 80 s (more samples will be loaded, it’s good because we use so little BigDye);

- Window>Status

- Click the Run button, note the date and time (you will need it to find your sequences later;

- It takes 1 h per sample.

5. Retrieve the data on the computer:

- Start “Sequencing Analysis 5.2” software

- User name = geneseq

- Password = geneseq

- File>Add sample: Choose your folder according to the date and time when your samples were run;

- Press OK, and my samples will be displayed;

- (Select all, click “Clear” button to make the window clear);

- Select: Dye Set Primer: KB_310_POP4_BDTV3_36std.mob (from pull down menu);

- Select: Matrix File: 310Matrix5.mtx (from pull down menu);

- Make selections for all samples (use the Ctrl-D trick as mentioned above);

- Check all boxes in front, and then hit the green Run button;

- Colors indicate quality: Blue = excellent, green = good, yellow = bad;

- Go to Sequence box, mark all, copy, paste in WordPad.