Wiki/Team:Warsaw/protocols

Culture E. coli producer strain in 10 ml of liquid LB medium for 8 hours. Then use it to inoculate 1000 ml of liquid LB medium with 0.5 mM IPTG and grow it overnight. In the morning spin down the culture (5000 RPM, 10 min, 4°C). Resuspend the pellet in PBS buffer and disrupt cells by sonication. Spin down sonication mixture (13200 RPM, 10 min, 4°C) and discard supernatant – protein is present in sonication debris. Resuspend it in sterile ice cold ddH2O and Spin down (13200 RPM, 10 min, 4°C). Discard supernatant and resuspend it in sterile ice cold ddH2O and store at 4°C.

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Purification of His_A_alpha

Culture, induce and disrupt E. coli in the same way as to purify His_Z_alpha. The protein is present in supernatant (about 10% of total protein) and can be added to selection medium without further purification. Nevertheless we purified it to determine how much exactly should be added:

1. Swing sonication products with Ni-nta-agarose bed for 2 hours at 4°C
2. Load them onto column
3. Wash the bed with 20 mM imidasole buffer
4. Elute purified protein with 100 mM imidasole

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Testing various hunter/prey combinations

1. Setup of culture E. coli carrying "hunter" with kanamycin and 0.2 mM IPTG
2. Inoculate liquid LB medium with kanamycin, 50 μg/ml ampicillin, 0.2 mM IPTG and "prey" (the control is medium without "prey")
3. Grow it 4-16h
4. Observe growth, or its lack
5. Isolate the plasmid DNA
6. Perform control digestion

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Plasmid DNA isolation

We use "Plasmid Mini" plasmid DNA isolation kit from A&A Biotechnology and follow the protocol of producer.

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DNA isolation from agarose gel

We use "Gel-Out" DNA isolation kit from A&A Biotechnology and follow the protocol of producer.

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DNA purification after enzymatic reaction

We use "Clean-Up" DNA purification kit from A&A Biotechnology and follow the protocol of producer.

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Genomic DNA isolation

We use "Genomic-Mini" universal genomic DNA isolation kit from A&A Biotechnology and follow the protocol of producer.

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DNA digest

We use restriction enzymes and buffers provided by Fermentas. Overall volume of digest mix is either 20 μl, either 50 μl in case of digesting for ligation. We usually use 1 μl of restriction enzyme and the buffer in 10x dilution (as they initially are 10x concentrated). The rest of mix is plasmid DNA.

Control digests are set up for 1 hour.
Digests for cloning take 3 hours or are left overnight.
Enzymes are deactivated by high temperature or by putting on gel, according to producer's recommendations.

Keep the bacteria on ice during the procedure. Pour ca. 25 ml of bacteria into a falcon tube and spin at 4°C at 4 krpm, 8 min with prolonged acceleration and deceleration. Remove supernatant. The pellet mustn't run dry. You can pour another portion of bacteria onto it and spin again. After desired amount of bacteria in pellet is collected, add CaCl₂ in an amount of 10% of initial culture used for spinning. Suspend the pellet until no debris is visible on the bottom. Incubate 45 min on ice. Then spin 8 min at 4 kg and remove supernatant. Suspend the pellet in 3 ml CaCl₂ and divide into aliquots of 100 μl.

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Preparation of electrocompetent bacteria

• Set up bacterial culture in 10 ml.
• Use the culture for inoculation of 1 L of medium and let it grow at 18°C until it reaches OD 0.6 - 0.8.
• Spin for 10 min at 6 krpm.
• Remove supernatant and suspend the pellet in 1 L of H₂O.
• Spin for 10 min at 6 krpm.
• Remove supernatant and suspend the pellet in 1 L of H₂O.
• Spin for 10 min at 6 krpm.
• Remove supernatant and suspend the pellet in 0.5 L of H₂O.
• Spin for 10 min at 6 krpm.
• Suspend the pellet in 20 ml 10% glycerol.
• Spin for 10 min at 6 krpm.
• Suspend the pellet in 3 ml 10% glycerol.
• Divide into aliquots of 40 μl and freeze in liquid nitrogen.

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Electrotransformation

• Pour 100 ml H₂O plus desired amount of DNA into electroporation cuvette.
• Add 40 ul of bacteria.
• Electroporate.
• Add 0.5 ml of LB.
• Incubate with shaking at 37°C.
• Plate.

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Chemotransformation

Add desired volume of DNA to the 100-μl-culture in eppendorf tube. Incubate 30 min on ice. Heat shock for 90 s at 42°C. Incubate 10 min on ice. Add 0.9 ml of culture medium and let the bacteria grow at 37°C.

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Ligation

We use the following mixture:

1. appropriate volumes of vector and insert DNA (usually concentration of insert 3X higher than that of vector)
2. 2 μl of ligation buffer
3. 1 μl of T4 DNA ligase (purchased from Fermentas)
4. nuclease-free water

Overall mix volume is 20 μl.
If ligated DNA has sticky ends - incubate 2h at room temperature; if ligated DNA has blunt ends - perform overnight incubation at 18°C.

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DNA ends blunting

Prepare digestion mix in overall volume of 50 μl.
Add to reaction mix:

• 1.5 μl of 2 mM dNTPs
• 0.5 μl Klenow fragment (for 5' sticky ends)
• 0.5 μl T4 DNA polymerase (for 3' sticky ends)
• Incubate overnight at 37 degrees.

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Standard concentrations of antibiotics and other supplements

Ampicillin
100 μg/ml for high copy number plasmids (pET15b)
30 μg/ml for one-copy plasmid (pZC320)
50 μg/ml for testing various hunter/prey combinations

Tetracycline
12-15 μg/ml

Kanamycin
30 μg/ml

Rifampicin
300 μg/ml

Chloramphenicol
35 μg/ml

X-Gal
40 μg/ml

IPTG
For liquid LB broth: variable concentration, depends on induced protein
For plates (blue-white screening): 0.1 mM

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Rifampicin test

1. Transform competent E. coli GM2163 or Top10 cells with:
   
   • pMPM-T5+AID
   • pMPM-T5+AID+T7 (transcriptional fusion)
   • pMPM-T5+AID-T7 (translational fusion)
   • pMPM-T5+AID+AID-T7
   • pMPM-T5+T7
   and plate on LB + Amp 30 μg/ml + Tet.


2. Inoculate two tubes with 3 ml LB + Amp30 μg/ml + Tet + 100 μl of 20% L-arabinose with colonies of transformants (negative control without arabinose)
3. Incubate overnight at 37°C
4. Plate 200 μl of bacterial culture on LB + 300 μg/ml Rifampicin + Tet
5. Incubate overnight at 37°C.

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Removing of 5' phosphate groups from DNA ends

1. Make digestion mix in overall volume of 50 μl.
2. Add 1 μl of Calf Intestinal Alkaline Phosphatase to the reaction mix.
3. Incubate 2h or overnight at 37°C.

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TAXI protocol (Tet+Ap 30+X-Gal+IPTG)

1. Single transformations of competent E. coli GM2163 (or TOP10 - in this case steps with GM2163 used were omitted, induced transformants were plated immediately on TAXI) carrying plasmid pZC320 with:
   
   • pMPM-T5+AID
   • pMPM-T5+AID+T7 (transcriptional fusion)
   • pMPM-T5+AID-T7 (translational fusion)
   • pMPM-T5+T7
2. Induction using of L-arabinose (100 μl 20% inductor/3 ml LB broth Ap 30 μg/ml + standard Tet) and negative control of each probe
3. Isolation of plasmids (pMPM-T5+AID, pMPM-T5+AID+T7 (transcriptional fusion) , pMPM-T5+AID-T7 (translational fusion) and pMPMT5+T7 )
4. Transformations of competent E. coli TOP10 with the isolated plasmids.
5. Plating on TAXI (Tet+Ap 30+X-Gal+IPTG)

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Protein concentration measurement (BCA method)

1. Place 10 μl of sample in a cuvette. Use 10 μl of solution, in which the sample is suspended, as control.
2. Mix BCA (bicinchoninic acid) with CuSO₄ (concentration?) at ratio of 50:1 .
3. Add 1.99 ml of BCA with CuSO₄ to the cuvetes.
4. Incubate 30 min at 37°C.
5. Measure absorbance at 562 nm.
6. Read protein concentration from reference curve.

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PCR

Most PCR was carried out in following conditions:
3 min 94°C - preincubation
30 s 94°C - melting
30 s annealing (temperature depends on primer sequence)
72°C (elongation time depends on length of product)
The above steps were repeated 15 - 35 times (depending on PCR efficiency)
5 min 72°C additional elongation
hold 4°C

PCR standard mix
For colony PCR (10 μl of reaction)
1 μl of each primer
1.2 μl MgCl₂ (25 mM)
0.8 μl dNTPs mix (2 mM)
1 μl Pfu buffer
0.2 μl Pfu turbo polymerase water up to 10 μl
template - bacterial cells suspended in PCR mix

To obtain PCR product for cloning (50 μl)
5 μl of each primer
6 μl MgCl₂ (25 mM)
4 μl dNTPs mix (2 mM)
5 μl Pfu buffer
1 μl Pfu turbo polymerase
template depends on DNA concentration
water up to 50 μl

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Polymerase Chain Ligation

It's PCR reaction on two partially complementing templates. In our project all fusions containing linkers were put together using this technique. Typical mix:
5 μl of each primer
6 μl MgCl₂ (25 mM)
1 μl dNTPs mix (10 mM)
5 μl Pfu buffer
1 μl Pfu turbo polymerase
achieving equal amounts of both templates is crucial
water up to 50 μl

Note about cycling conditions: It's very important to check that melting temperature of complementing region is lower than elongation temperature, thus in all of our PCL reactions elongation was carried out at 68°C