Conjugation protocol

Steps

1. Culture C.crescentus and Ecoli.S17-1 overnight with the liquid medium.When the OD of C.crescentus reaches 2 and S17-1’s reaches 0.5,we can stop cultivating.
2. Mix S17-1 and C.crescentus in a volume ratio of 2:1.Centrifuge it with 4000r/min and suspend the thallus again with 200μl LB liquid medium.
3. Spread out an 0.45μm nitrocellulose filter on the LB solid medium without antibiotics.
4. Spread the mixed bacteria liquid in step 2 onto the filter and incubate it at 30℃ for 3-7h.
5. After incubation,elute the thallus from the filter with 200-1000μl LB liquid medium.
6. Spread it on the PYE solid medium with antibiotics and incubate it at 30℃.Then we can get the C.crescentus with the target vector.

Conjugation of red light sensing-imaging system

We transfer the red and blue luminous plasmid following the conjugation protocol. [Pic][Pic]

Verification of C.crescentus

We use Gram Staining to verify the C.crescentus:

1. Prepare thin smear bacteria on a clean slide.
2. Cover the smear with few drops of crystal violet for 1-2min.
3. Wash excess strain with water.
4. Add few drops of iodine solution on the smear for 1-2min.
5. Rinse in water.
6. Wash with 95% ethanol for 20s.
7. Rinse in water.
8. Stain with 1% safranin for 2-3min.
9. Wash stain in excess water.
10. Look at it under a microscope.

Regular Protocols

Gel Electrophoresis

Standard 1% agarose gel

(Generally, 0.7%~2% agarose gel is widely used in lab)

1. Measure out 1g agarose in scale, and then put the sample into conical flask.
2. Add 100mL 1XTAE into conical flask, shaking evenly.
   (about 1xTAE: dilute the 50x TAE to 1x TAE: add 10mL 50x TAE and 490 mL double distilled water to form as a system for further use)
3. Plastic wrap can be used to pack the bottleneck.
4. Use microwave oven to heat the solution about 5mins until the agarose is all dissolved and the solution is boiling and clear. It would be better if we shake the flask again after heating 2 mins.(CAUTION: HOT! Please use wool gloves when picking and placing flask.)
5. Let the gel cool down about 2 mins.
6. Pour the gel into gel tray with the appropriate comb inserted in the side of tray and wait till the gel solified(It may costs about 10 mins). Then remove the comb
7. Put the gel into gel box with the 1XTAE just immerse the gel exactly. Please let passages stay towards the negative pole(Often black).
8. Carefully add samples with 6X loading buffer and marker into passages. Dosage is based on the depth and width of passages.
9. Set electrophoresis conditions: 90V 600mA and 30mins and then turn on the power.
10. Disconnect the electrodes.
11. Utilize the devices that contains UV light to observe the gel and analyze the result.
12. Save the result in the computer for further use.

Gel Extraction

Preparation:

1. Check out whether ethyl alcohol is added into Wash Solution.
2. Check out whether Buffer B2 has sediment.
3. Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

1. Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
2. Measure out the weight of tubes of gel.
3. Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
4. Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
5. After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
6. Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
7. Do the step 5 again.
8. Centrifuge the empty columns at 9000xg about 1min.
9. Open the columns and air them about 10 mins.
10. Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
11. Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

The protocol is based on SanPerp Column Type DNA Gel Extraction Kit.

PCR System Preparation and Conditions Setting

PCR system(set 50ul system as an example):

The amount of substance of premier is written on the EP tubes. Calculating the volume of 1XTE required for 10uM primer solution is required before the system preparation.

1. Template DNA xul as required.
2. Forward Primer(10 uM) about 1 to 2 ul. The final concentration will be 0.2 to 0.4 uM.
3. Reverse Primer(10 uM) about 1 to 2 ul. The final concentration will be 0.2 to 0.4 uM.
4. 5xTransStart FastPfu Fly Buffer 10 ul.
5. 2.5 mM dNTPs 5 uM. The final concentration will be 0.25 mM.
6. TransStart FastPfu Fly DNA Polymerase 1 ul.
7. Double distilled water added to 50 ul.

Reaction conditions

1. Calculate the Tm Value. If the complementary strip is smaller than 20 bp, Tm=4X(A+T)+2X(C+G), and the annealing temperature is Tm-5 degree centigrade. If not, use software to figure out the exact Tm value.(Also Tm value exist in report)
2. PCR cycle:
   
   1. 1 cycle of 95 degree centigrade about 2 min for pre-degeneration;
   2. 30 to 35 cycles of 95 degree centigrade about 10 sec, Tm-5 degree centigrade about 20 sec and 72 degree centigrade 1kb per min;
   3. 1 cycle of 75 degree centigrade about 5 mins and stay the device in 4 degree centigrade.

The protocol is based on TransStart FastPfu Fly DNA Polymerase.

Plasmid Extraction

Preparation

1. Check out whether RNaseA has been added in Buffer P1.
2. Check out whether ethyl alcohol has been added in Wash Solution
3. Check out whether sediment exist in Buffer P2 and P2.

Procedure

1. Absorb 1.5 to 5 mL bacteria solution in to EP tubes, centrifuge them at 8,000xg 2 mins and then discard the culture medium.
2. Add 250 ul Buffer P1 into sediment, and use spearhead to make bacteria suspended.
3. Add 250 ul Buffer P2, and overturn the EP tubes 5 to 10 times immediately and tenderly. Stand tubes about 2 to 4 mins.
4. Add 350 ul Buffer P3 and overturn the EP tubes 5 to 10 times again immediately and tenderly.
5. Centrifuge tubes at 12,000xg about 5 to 10 mins.
6. Pour the supernatant liquid into absorption columns ,centrifuge them at 8,000xg about 30 sec and then discard the liquid in the collecting pipe.
7. optional:Add 500 ul Buffer DW1 and centrifuge them at 9,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8. Add 500 ul Wash Solution, centrifuge them at 9,000xg about 30 sec and discard the liquid in the collecting pipe.
9. Do the step 8 again.
10. Centrifuge the empty columns at 9,000xg about 1 min.
11. Put the columns in clean 1.5mL EP tubes, add 50 to 100 ul Elution Buffer on the absorption film, stand 1 min and centrifuge about 1 min at 9,000xg.
12. Keep the DNA solution for further work.

The Protocol is based on SanPrep Column Type DNA Plasmid Extraction Kit.

PCR Production Extraction

Prepation

1. Check out whether ethyl alcohol has been added into Wash Solution.
2. Check out whether sediment exist in Buffer B3.
3. Check out whether isopropanol has been added into Buffer B3.

Procedure

1. Add Buffer B3 3 times volume of PCR system solution and incorporated thoroughly.
2. Centrifuge them at 8,000xg about 30 sec and drain the liquid in the collecting pipe.
3. Add 500 ul Wash Solution at 9,000xg about 30 sec and drain the liquid in the collecting pipe.
4. Do The step 3 again.
5. Centrifuge the empty pipes at 9,000Xg about 1 min.
6. Put the absorption column in some clean 1.5 mL EP tubes, add 15 to 40 ul Elution Buffer, stand for 1 min and centrifuge them at 9,000xg about 1min.
7. Keep the DNA solution.

Materials of LB Culture Medium

Liquid medium

Set a 1L LB medium as an example:

1. typtone 10g
2. Yeast Extract 5 g
3. NaCl 10 g

Solid medium

1. typtone 10g
2. Yeast Extract 5 g
3. NaCl 10 g
4. Agar A 15g

Pack medium with three layer of aluminium foil and rubber band．Then put the media into High-Pressure steam sterilization pot.

ATTENTION: Agar A, fine light yellow powder, is different from agarose, white powder.

Restriction Enzymes Analysis

Protocol

20ul system as following:

1. 2ul plasmid DNA sample;
2. 1ul restriction enzyme(both 1 ul for double digestion);
3. 2ul 10X green buffer;
4. double distilled water added to 20 ul.

Timetable:react the whole system at 37 degree centigrade about 2 hours and then inactivate the enzymes at 60 degree centigrade about 20 mins.

Preparation of Competent Cell

Procedure

1. Pick out the simple colony(DH5α) and cultivate bacteria in 2 to 3 mL LB media at 37 degree centigrade about 12 to 16 hours with shocking.
2. Take 0.05mL nutrient solution into 50 mL media and cultivate bacteria at 37 degree centigrade about 2 to 3 hours with shocking till the OD550 reaches 0.2 to 0.4.
3. Absorb 1.5mL nutrient solution into EP tubes with ice-bath about 10 mins.
4. Centrifuge the tubes at 4000xg at 4 degree centigrade about 10mins and discard the supernatant liquid.
5. Utilize 0.5 to 1 mL 0.1M calcium chloride with 15% glycerol to suspend bacteria.(*ATTENTION:USE SPEARHEAD TO SUSPEND BACTERIA TENDERLY INSTEAD OF VIBRATOR *)
6. Centrifuge the tubes at 4000xg at 4 degree centigrade about 8 mins and discard the supernatant liquid.
7. Utilize 100 ul 0.1M calcium chloride with 15% glycerol to suspend bacteria and then ready for transformation quickly or restore at -70 degree centigrade.(WITH ATTENTION AGAIN)

TIPS: EXPERIENCE SAYS MODERATE MAGNESIUM ION HELPS DEVELOPMENT OF COMPETENT CELL.

Transformation

NOTE:Generally, competent bacteria are restrored in -70 degree centrigrade environment.

Procedure

1. Take the competent bacteria from refrigerator and incubate them into ice about 5 mins until it is dissolved
2. Absorb 100pg to 10 ng plasmid(normally 1 to 2 uL, DO NOT add more than 5% volumn of bacteria solution) and mix it with bacteria solution thoroughly.
   ATTENTION: Please operate this step tenderly!!!
3. Put the tubes on the ice about 30 mins.(Time SHOULD BE ACCURATE)
4. Make a heat shock at 42 degree centigrade about 30 sec(TIME SHOULD BE ACCURATE)
5. Put the tubes on the ice about 2 to 3 mins again.
6. Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 40 to 60 min.
7. Centrifuge them at 12,000xg about 15 sec and we will se sediment in the tubes.
8. Discard the supernatant liquid and leave about 220 ul medium.
9. Coat plate: add 200 ul solution in a large plate while add 20 ul solution in a small plate.
10. Cultivate these bacteria overnight for further use.

Fluorescence Cultivation

1. Ingredient of M9 Medium:
   
   1. 99% 1x M9 solution including vitamin B1.
   2. 0.9% 50% glycerol.
   3. 0.2% 1mol/L magnesium sulfate.
   4. 0.01% 1mol/L calcium chloride.
2. For inducible promoter Plac:
   
   1. Select single colony from plate and inoculate it into 3 mL M9 medium, including chloramphenicol.
   2. Cultivate the bacteria in 37 degree centigrade at 200 rpm.
   3. Dilute the bacteria solution 1,000 times and then inoculate 3 mL into a new M9 medium.
   4. Cultivate the bacteria in 37 degree centigrade at 200 rpm about 4.5 hours until the OD 600 grow up to 0.15.
   5. Dilute IPTG into different concentration(every group keeps 3 tubes).
   6. Add IPTG into bacteria solution and measure the fluorescence expression. The OD value should be measured every 2 min.
   7. After 30 mins, measure the fluorescence intensity and OD 600 every 30 mins in 4 hours.