Wiki/Team:Warsaw/protocols

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Purification of His_Z_alpha and His_Z_omega

Culture E. coli producer strain in 3 ml of liquid LB medium + kanamycin for 8 hours. Then use it to inoculate 200 ml of liquid LB medium + kanamycin supplemented 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 – purified protein is present in sonication debris. Resuspend it in sterile ice cold ddH2O and store at 4°C.

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. Swinging of sonication products with Ni-nta-agarose bed for 2 hours at 4°C
  2. Loading onto column
  3. Washing of the bed with 20 mM imidasole buffer
  4. Elution with 100 mM imidasole

Mieżenie stężenia białka

metodą BCA 1. do kuwetek dodano po 10 mikrolitrów preparatu, a do kontroli 10 mikrolitrów rostworu w jakim jest preparat 2. zmieszano BCA (pełna nazwa?) z CuSO4 (stężenie?) w stosunku 50 do 1 3. dodano 1.99 ml tego roztworu do kuwetek 3. inkubowano 30 min w 37 stopniach C 4. mieżono absorbancję przy długości fali? 5. stęzenie bialka odczytywano z krzywej wzorcowej

Plasmid DNA isolation

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

DNA isolation from agarose gel

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

DNA purification after enzymatic reaction

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

Genomic DNA isolation

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

trawienia

najczęstrze trawienia: bamHi SacI ndei saci kpni psti - Bam buffer bamhi psti - Bam buffer xbaI psti ecori spei p>

Preparation of chemocompetent bacteria

Keep the bacteria on ice during the procedure. Pour ca. 25 ml of bacteria into a falcon tube and spin in 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.

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.

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.

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.

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 - incubation durates 2h in room temperature; if ligated DNA has blunt ends - overnight incubation in 18°C.

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 in 37 degrees.

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)

Tetracycline
12-15 μg/ml

Kanamycin
30 μg/ml

Rifampicin
300 μg/ml

X-Gal
40 μg/ml

IPTG
liquid LB broth: variable; depends of inducted protein
plates (blue-white screening): 0.1 mM