Team:St Andrews/Lab-book

From 2012.igem.org

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             <h1>Lab Book</h1>
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             <h1>Lab book</h1>
              
              
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<p>1.      Frozen cells were thawed from -80 °C.</p>
<p>1.      Frozen cells were thawed from -80 °C.</p>
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<p>2.      Thawed cells (10μL)were mixed with vector (1μL), and left on ice for 30 min.</p>
+
<p>2.      Thawed cells (10 μL) were mixed with vector (1 μL), and left on ice for 30 min.</p>
-
<p>3.      1 mL of LB liquid medium was warmed at 37°C for 30 min.</p>
+
<p>3.      LB liquid medium (1 mL) was warmed at 37°C for 30 min.</p>
<p>4.      The cell/vector mix was heated at 42 °C for 45 seconds.</p>
<p>4.      The cell/vector mix was heated at 42 °C for 45 seconds.</p>
-
<p>5.      Warm LB (50μL) was added to the cell vector mix.</p>
+
<p>5.      Warm LB (50 μL) was added to the cell vector mix.</p>
<p>6.      Mixture was shaken at 37 °C for 30 min.</p>
<p>6.      Mixture was shaken at 37 °C for 30 min.</p>
<p>7.      Mixture was plated onto ampicillin agar plates and left overnight.<p>
<p>7.      Mixture was plated onto ampicillin agar plates and left overnight.<p>
<p>8.      Recombinant <i>E. Coli</i> was harvested from agar plate by scratching gently with the scraper.</p>
<p>8.      Recombinant <i>E. Coli</i> was harvested from agar plate by scratching gently with the scraper.</p>
-
<p>9.      The scraper tip was placed into Falcon tubes with LB (10mL)and ampicillin (1 μL) to transfer the <i>E. Coli</i> into medium.</p>
+
<p>9.      The scraper tip was placed into Falcon tubes with LB (10 mL) and ampicillin (1 μL) to transfer the <i>E. Coli</i> into medium.</p>
<p>10.  The <i>E. Coli</i> was incubated in a shaker at 37 °C for less than 20 hr.</p>
<p>10.  The <i>E. Coli</i> was incubated in a shaker at 37 °C for less than 20 hr.</p>
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<p>1. <u>Preparing 1% agarose solution</u>: For 100 mL, dissolve 1 g agarose powder in 100 mL TAE (Tris base, acetic acid and EDTA) and heat in microwave. Then, add ethidium bromide (8 μL for 100 mL).</p>
+
<p>1. <u>Preparing 1% agarose solution</u>: Agarose powder (1 g) was dissolved in TAE (Tris base, acetic acid and EDTA) buffer (100 mL). This was heated in the microwave until the solution was transparent. Ethidium Bromide (8 μL) was added.</p>
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<p>2. Pour solution into plastic holder, add comb, and allow to cool - gel will set.</p>
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<p>2. The solution was poured into an assembled gel rigand allowed to set.</p>
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<p>3. Load samples into wells (5-20 μL), also adding DNA ladder.</p>
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<p>3. Prepared samples were loaded into the wells(5-20 μL) along with DNA ladder (5 μL).</p>
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<p>4. Run at 80 V for 20 min. to 50 min. depending on size of gel.</p>
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<p>4. The gel was run at 80 V for 20-50 min.</p>
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<p>5. Visualize under UV light.
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<p>5. The gel was visualised under UV light
<BR>&nbsp;<BR>
<BR>&nbsp;<BR>
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<p>6. Ratios for sample preparation vary depending on the type of sample:
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<p>Ratios for sample preparation vary depending on the type of sample:
<ul>
<ul>
<li>Miniprep - use 4 μL sample and 2 μL dye</li>
<li>Miniprep - use 4 μL sample and 2 μL dye</li>
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<p>1.      Prepare the sample in the following sequence (30μl):
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<p>1.      The sample (30μl) was prepared in the following sequnce:
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<li>20 μL miniprep</li>
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<li>Miniprep (20 μL)</li>
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<li>8 μL corresponding buffer</li>
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<li>Corresponding buffer (8 μL)</li>
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<li>1 μL of each restriction enzymes</li></p>
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<li>Each restriction enzyme (1 μL of each)</li></p>
<BR>&nbsp;</BR>
<BR>&nbsp;</BR>
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<p>2. Incubate in 37 °C water bath for 1 - 12 h, depending on brand of restriction enzyme.</p>
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<p>2. The sample was incubated at 37 °C in a water bath.  Timings depended on the restriction enzymes brand.</p>
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<div class="span2.5"><p><u>High-fidelity:</u>
<div class="span2.5"><p><u>High-fidelity:</u>
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<li>10 μL buffer</li>
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<li>buffer (10 μL )</li>
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<li>36 μL water</li>
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<li>water(36 μL )</li>
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<li>1 μL DNTP</li>
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<li>DNTP(1 μL)</li>
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<li>1 μL of each primers</li>
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<li>Forward primer (1 μL)</li>
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<li>1 μL DNA</li>
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<li>Reverse primer (1 μL)</li>
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To make a 50 μL solution, spin to mix, then add
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<li>DNA Template (1 μL)</li>
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<li>1 μL DNA polymerase</li></p></div>
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This is spun down to mix before adding
 +
<li>DNA polymerase (1 μL)</li></p></div>
<div class="span2.5"><p><u>Clontech</u>
<div class="span2.5"><p><u>Clontech</u>
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<li>22 μL water</li>
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<li>water (22 μL)</li>
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<li>25 μL buffer with dNTPs</li>
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<li>buffer with dNTPs (25 μL)</li>
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<li>1 μL of each primers</li>
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<li>Forward Primer (1 μL)</li>
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<li>1 μL DNA</li>
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<li>Reverse Primer (1 μL)</li>
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To make a 50 μL solution, spin to mix, then add
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<li>DNA(1 μL )</li>
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<li>1 μL DNA polymerase</li></p></div>
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This is spun down to mix before adding
 +
<li>DNA polymerase (1 μL)</li></p></div>
<div class="span2.5"><p><u>KOD</u>
<div class="span2.5"><p><u>KOD</u>
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<li>35 μl water</li>
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<li>water (35 μl)</li>
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<li>5 μL 10x buffer</li>
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<li>10x buffer (5 μL)</li>
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<li>5 μL dNTPs</li>
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<li>dNTPs(5 μL)</li>
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<li>5 μL MgSO<sub>4</sub></li>
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<li>MgSO<sub>4</sub>(5 μL)</li>
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<li>1 μL of each primers</li>
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<li>Forward Primer (1 μL)</li>
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<li>1 μL DNA</li>
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<li>Reverse Primer (1 μL)</li>
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To make a 50 μL solution, spin to mix, then add
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<li>DNA (1 μL)</li>
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<li>1 μL DNA polymerase</p></div>
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This is spun down to mix before adding
 +
<li>DNA polymerase(1 μL)</p></div>
<div class="span2.5"><p><u>GoTaq</u>
<div class="span2.5"><p><u>GoTaq</u>
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<li>23 μL water</li>
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<li>water (23 μL)</li>
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<li>25 μL GoTaq Master Mix buffer</li>
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<li>GoTaq Master Mix buffer (25 μL)</li>
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<li>1 μL of each primer</li>
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<li>Forward primer (1 μL)</li>
 +
<li>Reverse primer (1 μL)</li>
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                   <strong>Colony PCR</strong> (with GoTaq)
                   <strong>Colony PCR</strong> (with GoTaq)
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<p>1. Prepare multiple PCR tubes for each plate and make a solution as follows:
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<p>1. PCR tubes were prepared in the following sequence:
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     <li>23 μL water </li>
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     <li>Water (23 μL) </li>
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     <li>25 μL GoTaq® MasterMix buffer</li>
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     <li>GoTaq® MasterMix buffer (23 μL)</li>
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     <li>1 μL of each primers</li>
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     <li>Forward and reveerse primers (1 μL each)</li>
     <p>to make a 50μL PCR solution</p></p>
     <p>to make a 50μL PCR solution</p></p>
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<p>2. Prepare and label corresponding Eppendorf tubes for each PCR tube</p>
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<p>2. An Eppendorf tube was prepared for each PRC tube.</p>
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<p>3. Using a small pipette tip, scratch one colony off a plate and dip the tip into the PCR tube. Store the tip in the corresponding Eppendorf tubes (for further incubation, if positive).</p>
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<p>3. Using a small pipette tip, one colony was scratched off a plate and dipped into the tip into the PCR tube. Tips were then stored in the corresponding Eppendorf tubes (for further incubation, if positive).</p>
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<p>4. Run PCR using optimal annealing temperature for the required gene, and then take samples of the PCR products for electrophoresis.</p>
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<p>4. The PCR was ran using optimal annealing temperatures for the required gene, and then the samples of the PCR run straight on electrophoresis.</p>
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<p>6.  Select the positive from all samples and incubate the corresponding tips.</p>
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<p>6.  Positive results from the samples were incubated from the corresponding tips.</p>
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                  <strong>Phosphatase treatment of linearized vector</strong>
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<p>1. Add phosphatase buffer to sample in accordance with its concentration factor.</p>
 +
<p>2. Add 1μL phosphatase.</p>
 +
<p>3. Incubate 60 mins at 37°C. </p>
 +
<p>4. Heat-shock for 5 mins at 65°C. </p>
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<p><u>Ligation molar ratio calculation</u>:</p>
<p><u>Ligation molar ratio calculation</u>:</p>
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<p>Compare UV light exposure intensity for quantity in ng (i.e. the concentration of both insert and vector) against ladder position for sequence length (i.e. molar weight ratio of the two)
+
<p>
-
Rule of 3: three times as much insert as vector ensures successful ligation results</p>
+
Exposure intensity from UV light was used to quantify the DNA in ng (i.e. the concentration of both insert and vector) against ladder position for sequence length (i.e. molar weight ratio of the two)
 +
<br>Rule of 3: Three times as much insert as vector ensures successful ligation results</br></p>
<BR>&nbsp;<BR>
<BR>&nbsp;<BR>
-
<p>1. Prepare 20 μL of ligation solution</p>
+
<p>1. Ligation solution (20 μL) was prepared</p>
-
           <ul><li>2 μL 10x buffer
+
           <ul><li>10x buffer (2 μL)
-
           <li>1 μL T4 ligase</li>
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           <li>T4 ligase (2 μL)</li>
-
           <li> ~17 μL consist of  vectors and insert, according to calculated ratio</li>
+
           <li> Vectors and insert (~17 μL), according to calculated ratio</li>
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           <li> make up to 20 μL using water</li></ul>
+
           <li> make up to a total of 20 μL using water</li></ul>
-
<p>2. Let stand at room temperature for 10-30 min.</p>
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<p>2. The ligation mixture was allowed to stand at room temperature for 10-30 minutes..</p>
-
<p>3. Mix the ligase-treated vector-insert mixture with 60 μl <i>E. Coli</i> (DH5-α), then proceed the standard transformation protocol and shake for 1 hr; finally add 200 μL LB liquid media.</p>
+
<p>3. The ligase-treated vector-insert mixture was mixed with <i>E. Coli</i> (DH5-α)(60 μL), then the standard transformation protocol was followed with shaking for 1 hr; finally LB (200 μL) was added.</p>
-
<p>4. Spread all contents of the shaken mixture onto agar plates, with an additional control of transformed <i>E. Coli</i> with non-inserted linearised vector.  Incubate overnight at 37 °C.</p>
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p>
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<p>1. Using LB as a blank reference, measure the optimal density of 1 mL of sample in the photometer.</p>
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<p>1. The optimal density (OD) of a portion of the sample (1 mL) was measured in the spectrophotometer using LB as a blank.</p>
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<p>2. For induction, the OD should be at 0.5. The sample can be grown further or diluted if necessary.</p>
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<p>2. If the OD was approximately 0.5 the sample was induced. The sample could be incubated further or diluted if necessary.</p>
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<p>2. IPTG should be added to a concentration of 1 mM. This figure will vary if protein expression/cell growth is carried out at a lower non-standard temperatures to accomodate for the slower metabolism.</p>
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<p>2. IPTG (1 mM) was added.</p>
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<p>1. Spin down the <i>E. Coli</i> in a centrifuge.</p>
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<p>1. The <i>E. Coli</i> was spun down in a centrifuge.</p>
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<p>2. Discard the supernatant and add in 1 mL of PBS (phosphate buffer saline) to resuspend the pellet, then tansfer to Eppendorf tubes.</p>
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<p>2. The supernatant was discarded and phosphate buffer saline(PBS, 1 mL) was added to resuspend the pellet before the mix was transferred to an Eppendorf tube.</p>
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<p>3. Spin down the <i>E. Coli</i> at full speed for 1 min., discard supernatant and then put on ice/in cool room for storage.</p>
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<p>3. The <i>E. Coli</i> was spun at full speed for 1 min., the supernatant discarded and the pellet was put on ice for storage.</p>
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<p>4. Prepare lysozyme solution (10 mg/mL) in lysis buffer.</p>
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<p>4. Lysozyme solution (10 mg/mL) was prepared in lysis buffer.</p>
-
<p>5. Add 200 μL into each pellet and resuspend it; vortex and shaker incubate at 37 °C for 30 min. to 1 h.</p>
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<p>5. Lysozyme solution (200 μL) was added to each pellet and they were resuspended and vortexed before being incubated in a shaker (37 °C) for 30 min. to 1 h.</p>
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<p>6. Sonicate the cells.</p>
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<p>6. The cells were sonicated.</p>
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<p>7. Take 10 μL total sample and add in 10 μL 2x dye.</p>
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<p>7. Total sample (10 μL) was removed and 2x dye (10 μL) was added.</p>
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<p>8. Spin the original tubes for 1 min. at full speed, remove the supernatant, then resuspend the mass with 200 μL PBS.</p>
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<p>8. \the original tubes were spun for 1 min. at full speed and the supernatant was removed, the mass was resuspended with PBS (200 μL).</p>
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<p>9. Take 10 μL membrane sample from the resuspended solution and add in 10 μL 2x dye.</p>
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<p>9. Membrane sample (10 μL) was taken from the resuspended solution and 2x dye (10 μL) was added.</p>
-
<p>10. Denature the protein samples on the heating block at 95° C or 75°C (for membrane protein).</p>
+
<p>10. The protein samples were denatures on a heating block (95° C or 75°C for membrane protein).</p>
-
<p>11. Cool on ice for 1 min., then load the samples to the gel (prepared according to <a gref="http://tryps.rockefeller.edu/Protocols/western_blot_vm.pdf">SDS-PAGE standard protocol</a>) and run at 150 V for around 45 min.</p>
+
<p>11. The samples were cooled on ice for 1 min., then loaded on to the protein gel (prepared according to <a gref="http://tryps.rockefeller.edu/Protocols/western_blot_vm.pdf">SDS-PAGE standard protocol</a>) and run at 150 V for around 45 min.</p>
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<p>1. Making the membrane transfer sandwiches: 2x A2 at the bottom, then 2x A2.</p>
<p>1. Making the membrane transfer sandwiches: 2x A2 at the bottom, then 2x A2.</p>
-
<p>2. Cut out the membrane, GE Healthcare HyBond ECL films, using a glass template; put the membrane onto moist A2 filter papers and press flat using a roll; cut off excessive margins if necessary.</p>
+
<p>2. The membrane, GE Healthcare HyBond ECL films, was cut out using a glass template; the membrane was put onto moist A2 filter papers and pressed flat using a roll.</p>
-
<p>3. Open the gel slides using a wedge; remove the thinner slide and, using the wedge, prize the protein gel from the thicker plate and onto the membrane.</p>
+
<p>3. The gel slides were opened using a wedge; removed the thinner slide and, using the wedge, prized the protein gel from the thicker plate and onto the membrane.</p>
-
<p>4. Gently press out air bubbles between the gel and membrane.</p>
+
<p>4. The airbubbles were gently pressed out between the membrane and the gel.</p>
-
<p>5. Add on CAT filter paper.</p>
+
<p>5. CAT filter paper was added.</p>
-
<p>6. Run the machine for 1hr at constant ampere of 40 mA for each gel.</p>
+
<p>6. The power pack was run for 1hr (40 mA).</p>
-
<p>7. If the rpestained markers have transferred, place the CAT filter papers onto a rocker and add stain. If using Ponceau, the stain can be reused.</p>
+
<p>7. If the restained markers have transferred, place the CAT filter papers onto a rocker and add stain. If using Ponceau, the stain can be reused.</p>
-
<p>8. Rinse with water to destain.</p>
+
<p>8. Destain was done by rinsing with water.</p>
-
<p>21. Pour in 50 mL 5% milk powder and leave on rocker for 2 hours at room temperature; if leaving overnight, rock in cold room using lower speed.</p>
+
<p>21. Milk powder (50 mL 5%) was added and left to rock for 2 hours at room temperature.  If leaving overnight, rock in cold room using lower speed.</p>
-
<p>9. Decant blocking milk solution, and then add in 10 mL 1% milk with 5μl of diluted antibodies. Rock for 1 h.</p>
+
<p>9. The blocking milk solution was decanted, and replaced with milk (10 mL 1%) with antibodies (5μl of diluted). This was rocked for 1 h.</p>
-
<p>10. Pour off the milk-antibody solution and wash with 0.3% PBS and Tween, three times in 10 min. intervals.</p>
+
<p>10. The milk-antibody solution was poured off and washed with PBS (0.3%) and Tween, three times at 10 min intervals.</p>
-
<p>11. Prepare a solution of "Supersignal west Fermto trail" kit (1:1 ratio, 1 mL per membrane).</p>
+
<p>11. A solution of "Supersignal west Fermto trail" kit (1:1 ratio, 1 mL per membrane).</p>
-
<p>12. Moisten membrane with visualization solution, then cover for 5 min. Then, dry off excess liquid.</p>
+
<p>12. The membrane was made moist with the visualization solution, covered for 5 min, then the excess liquid was removed.</p>
-
<p>13. Develop the membranes in the dark room.</p>
+
<p>13. The membranes were developed in a dark room.</p>
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<p>1. Spin down the <i>E. Coli</i> in a centrifuge.</p>
+
<p>1. The <i>E. Coli</i> was spun down in a centrifuge(800g, 10min).</p>
-
<p>2. Discard the supernatant and add in 1 mL of PBS to resuspend the pellet, then transfer to Eppendorf tubes.</p>
+
<p>2. With the supernatant discarded, PBS (1 mL) was used to resuspend the pellet, then transferred to Eppendorf tubes.</p>
-
<p>3. Spin down the <i>E. Coli</i> again at full speed for 1 min. and discard supernatant.</p>
+
<p>3. The <i>E. Coli</i> was spun again at full speed for 1 min. and the supernatant discarded.</p>
-
<p>4. Resuspend the pellet with 100 μL PBS and then transfer to glass tubes.</p>
+
<p>4. The pellet was resuspended using PBS (100 μL) and then transferred to glass sample vials.</p>
-
<p>5. Add organic solution for lysis, chloroform:methanol (1:2, 375 μL).</p>
+
<p>5. An organic mixture of chloroform:methanol (1:2(v/v), 375 μL) was added for lysis.</p>
-
<p>6. Vortex in the cold room for 1 h and then add 125 μL chloroform and 125 μL water to allow phase separation (lower organic layer contains lipids); let stand for at least 10 min.</p>
+
<p>6. The sample was vortexed for 1 h in the cold room, with chloroform (125 μL) and water (125 μL) added to allow phase separation (lower organic layer contains lipids); and left to stand for at least 10 min.</p>
-
<p>7. Spin down using special adapters at 3000 rpm for 5 min.</p>
+
<p>7. The sample vials were spun at 1000g at room temperature for 5 min.</p>
-
<p>8. Pipette the bottom layer into a glass sample tube, being careful not to take any milky layer.</p>
+
<p>8. The lower organic layer was transfered to a new glass vial avoiding the upper aqueous layer at all costs.</p>
-
<p>9. Dry the organic solvent under nitrogen and store.</p>
+
<p>9. The resultant lower phase lipid extract was then dried under N<sub>2</sub>, and the fatty acid stored in the fridge.</p>
 +
<p>9. The samples can be re-dissolved in chloroform:methanol (1:2) and are ready for Electrospray-mass spectrometry analysis for phospholipids and can be further processed for fatty acid derivation and analysis by Gas Chromatography-Mass Spectrometry.</p>
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               </div>
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<BR>&nbsp;<BR>
<BR>&nbsp;<BR>
<p>2. After labelling all screw-cap Eppendorfs, add the following assay components to each B and C tube:
<p>2. After labelling all screw-cap Eppendorfs, add the following assay components to each B and C tube:
-
     <ul><li>20 μL of 40 μM tricine buffer, made up to pH 8 with KOH</li>
+
     <ul><li> tricine buffer (20 μL, 40 μM), made up to pH 8 with KOH</li>
-
     <li>20 μL of 10 μM MgCl<sub>2</sub></li>
+
     <li>MgCl<sub>2</sub> (20 μL of 10 μM)</li>
-
     <li>10 μL ferredoxin</li>
+
     <li>Ferredoxin (10 μL)</li>
-
     <li>10 μL of 5μM NADPH</li>
+
     <li>NADPH (10 μL, 5μM)</li>
-
     <li>30 μL of catalase</li>
+
     <li>Catalase (30 μL)</li>
-
     <li>10 μL NADPH<sup>+</sup></li></ul></p>
+
     <li>NADPH<sup>+</sup> (10 μL)</li></ul></p>
<BR>&nbsp;<BR>
<BR>&nbsp;<BR>
-
<p>3. Add 100 μL of membrane sample, prepared as in the lipid extration. Use 500 μL lysozyme, and resuspend in tricine buffer. Run a <a href="http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html">Bradford assay</a> to determine protein content.</p>
+
<p>3. The membrane sample (100 μL), prepare
-
<p>4. Vortex at 4°C for 2 min.</p>
+
 
-
<p>5. Leave in water-bath at 37°C.</p>
+
 
-
<p>6. Add 250 μL chloroform and 250 μL water to stop assay reaction and allow for phase separation.</p>
+
 
-
<p>7. Samples will be used to carry out mass spec analysis.</p>
+
d as in the lipid extraction, was added to the mixture. Lysozyme (500 μL) was added, and resuspended in tricine buffer. The <a href="http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html">Bradford assay</a> was run to determine protein content.</p>
 +
<p>4. The sample was vortexed at 4°C for 2 min.</p>
 +
<p>5. It was put in a 37°C water bath.</p>
 +
<p>6. Chloroform (250 μL) and water (250 μL) inhibited the assay reaction and allowed for phase separation.</p>
 +
<p>7. Samples were used to carry out mass spec analysis.</p>
         </div>
         </div>
               </div>
               </div>
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                 <div class="accordion-inner">
                 <div class="accordion-inner">
-
<p>1. Aliquots of the lipd extract were transferred to 2mL glass vessels and dried under
+
<p>1. Aliquots of the lipid extract were transferred to 2mL glass vessels and dried under
nitrogen.</p>
nitrogen.</p>
-
<p>2. Base hydrolysis to release fatty acids were released by base hydrolysis using
+
<p>2. Base hydrolysis to release fatty acids was done using of concentrated ammonia (500 µL) and 50% propan-1-ol (50%) 1:1, followed by incubation for
-
500 µL of concentrated ammonia and 5|0% propan-1-ol (1:1), followed by incubation for
+
5 h at 50 °C.</p>
5 h at 50 °C.</p>
-
<p>3. After cooling, the samples are evaporated to dryness with nitrogen and dried twice more
+
<p>3. After cooling, the samples were evaporated to dryness with nitrogen and were dried twice more from methanol : water (1:1) (200 µL) to remove all traces of ammonia.</p>
-
from 200ul of methanol : water (1:1) to remove all traces of ammonia.</p>
+
<p>4. The protonated fatty acids are then extracted by partitioning between HCl (500 µL, 20 mM) and of ether (500 µL); the aqueous phase was re-extracted with fresh ether (500µL) and the combined ether phases were dried under nitrogen in a glass tube.</p>
-
<p>4. The protonated fatty acids are now extracted by partitioning between 500µL of 20mM HCl and 500uL of ether, the aqueous phase is re-extracted with fresh ether (500µL) and the combined ether
+
<p>5. The fatty acids were converted to fatty acid methyl esters (FAMEs), by adding diazomethane (3x 20 µL aliquots) to the dried residue, while on ice. After 30min, the samples were allowed to warm to RT and left to evaporate to dryness in a fume hood. </p>
-
phases are dried under nitrogen in a glass tube.</p>
+
<p>6. The FAME products were dissolved in dichloromethane (10-20 µL) and analysed (1-2 µL) by GC-MS on a Agilent Technologies (GC-6890N, MS detector-5973) with a ZB-5 column (30M x 25mm x 25mm, Phenomenex), with a temperature program of at 70 °C for 10min followed by a gradient to 220 °C at 5 °C /min and held at 220 °C for a further 15min </p>
-
<p>5. The fatty acids are converted to methyl esters (FAME), by adding diazomethane (3x 20uL
+
<p>7. Mass spectra were acquired from 50-500 amu. The identity of FAMEs was carried out by comparison of the retention time and fragmentation pattern with a Bacterial FAME standard (Supelco). </p> </div>
-
aliquots) to the dried residue, while on ice. After 30min, the samples were allowed to
+
-
warm to RT and left to evaporate to dryness in a fume hood. </p>
+
-
<p>6. The FAME products are dissolved in 10-20µL dichloromethane and 1-2µL analysed by
+
-
GC-MS on a Agilent Technologies (GC-6890N, MS detector-5973) with a ZB-5 column
+
-
(30M x 25mm x 25mm, Phenomenex), with a temperature program of at 70 °C for 10min
+
-
followed by a gradient to 220 °C at 5 °C /min and held at 220 °C for a further 15min.</p>
+
-
<p>7. Mass spectra were acquired from 50-500 amu. The identity of FAMEs was carried out
+
-
by comparison of the retention time and fragmentation pattern with a Bacterial FAME
+
-
standard (Supelco). </p> </div>
+
               </div>
               </div>
           </div>
           </div>
 +
<div class="accordion-group">
 +
              <div class="accordion-heading">
 +
                <a class="accordion-toggle" data-toggle="collapse" data-parent="#accordion2" href="#collapse19">
 +
                  <strong>Bradford Protein Assay</strong>
 +
                </a>
 +
              </div>
 +
              <div id="collapse19" class="accordion-body collapse" style="height: 0px; ">
 +
                <div class="accordion-inner">
 +
<p>The Bradford protein assay was carried out in accordance with standard procedure as described in BRADFORD, M., 1976. <a href=http://www.ciens.ucv.ve:8080/generador/sites/lab-bioq-gen/archivos/Bradford%201976.pdf>A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem, 7;72:248-54.</p>
 +
      </div>
 +
              </div>
 +
          </div>
 +
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-
<p>1. Spin down the E. coli in a centrifuge.</p>
+
<p>1. The <i>E. coli</i> was spun down in a centrifuge.</p>
-
<p>2. Discard the supernatant and add in 1 mL of PBS (phosphate buffer saline) to re-suspend the pellet. Transfer to Eppendorf tubes.</p>
+
<p>2. The supernatant was discarded and phosphate buffer saline (PBS, 1 mL) was added to re-suspend the pellet. The mixture was transferred to eppendorf tubes.</p>
-
<p>3. Spin down the E. coli at full speed for 1 min. Discard supernatant and then put on ice for storage.</p>
+
<p>3. The <i>E. coli</i> was spun down at full speed for 1 min. The supernatant was discarded and then put on ice for storage.</p>
-
<p>4. Prepare lysozyme solution (10 mg/mL) in lysis buffer.</p>
+
<p>4. Lysozyme solution (10 mg/mL) was prepared in lysis buffer.</p>
-
<p>5. Add 200 μL lysozyme solution to each pellet and re-suspend. Add 1 µL of benzonase nuclease, dissolve 1 protease inhibitor cocktail pill; vortex and incubate in shaker at 37 °C for 30 min. to 1 h.</p>
+
<p>5. Lysozyme solution (200 μL) was added to each pellet and re-suspended. Benzonase nuclease (1 µL) was added and 1 protease inhibitor cocktail pill was disolved in the solution; this was vortexed and incubated in a shaker at 37 °C for 30 min. to 1 h.</p>
-
<p>6. Sonicate the cells.</p>
+
<p>6. The cells were sonicated.</p>
-
<p>7. Spin down sonicated cells at full speed for 1 min in centrifuge.</p>
+
<p>7. The sonicated cells were spun down at full speed for 1 min in a centrifuge.</p>
-
<p>8. Prepare Nickel beads. Take 100 µL of Ni-NTA slurry per sample. Spin in centrifuge at full speed, remove supernatant. Add 100 µL of PBS per sample, re-suspend and spin down at full speed for 1 min.</p>
+
<p>8. The Nickel beads were prepared. Ni-NTA slurry per sample(100 µL). This was spun in centrifuge at full speed, and the supernatant removed . PBS was added (100 µL), the pellet was re-suspended and spun down at full speed for 1 min.</p>
-
<p>9. Remove supernatant; add 100 µL of PBS per sample, re-suspend and transfer to eppendorf tubes 100 µL per sample.Spin down at full speed for 1 minute in centrifuge, remove supernatant. Add supernatant of sonicated cells and re-suspend.</p>
+
<p>9. The supernatant was removed; PBS was added (100 µL), the pellet was re-suspended and transferred to eppendorf tubes (100 µL). This was spun down at full speed for 1 minute in a centrifuge,and the supernatant was removed. The supernatant of the sonicated cells was added and the mix was re suspended.</p>
-
<p>10. Agitate samples for 30 min to 3 h in cold room.Spin down samples at full speed for 1 minute in centrifuge and remove supernatant.</p>
+
<p>10. The samples were agitated for 30 min to 3 h in the cold room. The samples were spun down at full speed for 1 minute in centrifuge and the supernatant was removed.</p>
-
<p>11. Add 100 µL of NINTA elution buffer per sample; agitate samples for 10 to 30 min in cold room.</p>
+
<p>11. NINTA elution buffer (100 µL) was added and samples were agitated for 10 to 30 min. in the cold room.</p>
-
<p>12. Spin down samples at full speed for 1 min. Take 10 µL of supernatant and add 10µL 2x dye.</p>
+
<p>12. The samples were spun down at full speed for 1 min. and 10 µL of supernatant was removed.2x dye was added to the supernatant (10 µL).</p>
-
<p>13. Denature the protein samples on the heating block at 95° C.</p>
+
<p>13. The protein samples were denatures on the heating block at 95° C.</p>
-
<p>14. Cool on ice for 1 min., then load the samples to the gel (prepared according to SDS-PAGE standard protocol) and run at 150 V for 45 min.</p>
+
<p>14. The samples were cooled on ice for 1 min., and then loaded to the gel (prepared according to SDS-PAGE standard protocol) and run at 150 V for 45 min.</p>
</div>
</div>
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                 <div class="accordion-inner">
                 <div class="accordion-inner">
-
<p>1. Spin down the <i>E. coli</i> in a centrifuge.</p>
+
<p>1. The <i>E. coli</i> were spun down in a centrifuge.</p>
-
<p>2. Discard the supernatant and add in 1 mL of PBS (phosphate buffer saline) to re-suspend the pellet. Transfer to Eppendorf tubes.</p>
+
<p>2. The supernatant was discarded and phosphate buffer saline (PBS) (1 mL) was added to re-suspend the pellet. The mixture was transferred to eppendorf tubes.</p>
-
<p>3. The <i> E. coli</i> was spun down at full speed for 1 min. The supernatant was then discarded and stored on ice.</p>
+
<p>3. The <i> E. coli</i> in the tubes were spun down at full speed for 1 min. The supernatant was then discarded and stored on ice.</p>
<p>4. The lysozyme solution (10 mg/mL) was prepared in lysis buffer. </p>
<p>4. The lysozyme solution (10 mg/mL) was prepared in lysis buffer. </p>
-
<p>5. The lysozyme solution (200 μL) was added to each pellet and re-suspend. Benzonase nuclease (1 μL) was added, and one protease inhibitor cocktail pill was dissolved in the mixture. This was then
+
<p>5. The lysozyme solution (200 μL) was added to each pellet and re-suspended. Benzonase nuclease (1 μL) was added, and one protease inhibitor cocktail pill was dissolved in the mixture. This was then
vortexed and incubated in the shaker (37 °C for 30 min to 1 h). </p>
vortexed and incubated in the shaker (37 °C for 30 min to 1 h). </p>
<p>6. The cells were then sonicated. </p>
<p>6. The cells were then sonicated. </p>
-
<p>7. The sonicated cells were then spun down in the centrifuge at full speed for one minute. </p>
+
<p>7. The sonicated cells were then spun down at full speed for 1 min in the centrifuge . </p>
-
<p>8. GST beads were prepared. GST slurry (100 µL) was added per sample and spun down at full
+
<p>8. GST beads were prepared. GST slurry (100 µL) was added to each sample and spun down at full
-
speed. The supernatant was then removed. PBS (100 µL) was added per sample, and the mixture re-
+
speed. The supernatant was then removed. PBS (100 µL) was added to each sample, and the mixture re-
suspended and spun down at full speed for 1 min. </p>
suspended and spun down at full speed for 1 min. </p>
<p>9. The supernatant was then removed and PBS (100 µL) added per sample, re-suspended and
<p>9. The supernatant was then removed and PBS (100 µL) added per sample, re-suspended and
-
transferred to eppendorf tubes, 100 µL per sample. The mixture re-suspended and spun down at
+
transferred to eppendorf tubes, 100 µL per sample. The mixture spun down at
full speed for 1 min and supernatant was then removed. The supernatant of the sonicated cells was
full speed for 1 min and supernatant was then removed. The supernatant of the sonicated cells was
then added. </p>
then added. </p>
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Latest revision as of 02:56, 27 September 2012

Lab book

Protocols

1. Frozen cells were thawed from -80 °C.

2. Thawed cells (10 μL) were mixed with vector (1 μL), and left on ice for 30 min.

3. LB liquid medium (1 mL) was warmed at 37°C for 30 min.

4. The cell/vector mix was heated at 42 °C for 45 seconds.

5. Warm LB (50 μL) was added to the cell vector mix.

6. Mixture was shaken at 37 °C for 30 min.

7. Mixture was plated onto ampicillin agar plates and left overnight.

8. Recombinant E. Coli was harvested from agar plate by scratching gently with the scraper.

9. The scraper tip was placed into Falcon tubes with LB (10 mL) and ampicillin (1 μL) to transfer the E. Coli into medium.

10. The E. Coli was incubated in a shaker at 37 °C for less than 20 hr.

We used Qiagen's QIAprep Spin Miniprep Kit, the protocol can be found here.

1. Preparing 1% agarose solution: Agarose powder (1 g) was dissolved in TAE (Tris base, acetic acid and EDTA) buffer (100 mL). This was heated in the microwave until the solution was transparent. Ethidium Bromide (8 μL) was added.

2. The solution was poured into an assembled gel rigand allowed to set.

3. Prepared samples were loaded into the wells(5-20 μL) along with DNA ladder (5 μL).

4. The gel was run at 80 V for 20-50 min.

5. The gel was visualised under UV light
 

Ratios for sample preparation vary depending on the type of sample:

  • Miniprep - use 4 μL sample and 2 μL dye
  • Digestion - use 1 μL sample and 2 μL dye
  • PCR- use 10μl sample and 1μl dye

When using GoTaq®, simply load 10 μL onto the gel, as dye is already included.

Always make up to 10 μL using water.

1. The sample (30μl) was prepared in the following sequnce:

  • Miniprep (20 μL)
  • Corresponding buffer (8 μL)
  • Each restriction enzyme (1 μL of each)

  •  

    2. The sample was incubated at 37 °C in a water bath. Timings depended on the restriction enzymes brand.

    1. Prepare 20 μM primer working stock for both forward and reverse primers

    2. Prepare PCR according to polymerase brand:

    High-fidelity:

  • buffer (10 μL )
  • water(36 μL )
  • DNTP(1 μL)
  • Forward primer (1 μL)
  • Reverse primer (1 μL)
  • DNA Template (1 μL)
  • This is spun down to mix before adding
  • DNA polymerase (1 μL)
  • Clontech

  • water (22 μL)
  • buffer with dNTPs (25 μL)
  • Forward Primer (1 μL)
  • Reverse Primer (1 μL)
  • DNA(1 μL )
  • This is spun down to mix before adding
  • DNA polymerase (1 μL)
  • KOD

  • water (35 μl)
  • 10x buffer (5 μL)
  • dNTPs(5 μL)
  • MgSO4(5 μL)
  • Forward Primer (1 μL)
  • Reverse Primer (1 μL)
  • DNA (1 μL)
  • This is spun down to mix before adding
  • DNA polymerase(1 μL)

  • GoTaq

  • water (23 μL)
  • GoTaq Master Mix buffer (25 μL)
  • Forward primer (1 μL)
  • Reverse primer (1 μL)
  • We used Epoch BioLab's GenCatch PCR Cleanup Kit (Protocol) and Qiagen's QIAquick Gel Extraction Kit (Protocol).

    1. PCR tubes were prepared in the following sequence:

  • Water (23 μL)
  • GoTaq® MasterMix buffer (23 μL)
  • Forward and reveerse primers (1 μL each)
  • to make a 50μL PCR solution

    2. An Eppendorf tube was prepared for each PRC tube.

    3. Using a small pipette tip, one colony was scratched off a plate and dipped into the tip into the PCR tube. Tips were then stored in the corresponding Eppendorf tubes (for further incubation, if positive).

    4. The PCR was ran using optimal annealing temperatures for the required gene, and then the samples of the PCR run straight on electrophoresis.

    6. Positive results from the samples were incubated from the corresponding tips.

    1. Add phosphatase buffer to sample in accordance with its concentration factor.

    2. Add 1μL phosphatase.

    3. Incubate 60 mins at 37°C.

    4. Heat-shock for 5 mins at 65°C.

    Ligation molar ratio calculation:

    Exposure intensity from UV light was used to quantify the DNA in ng (i.e. the concentration of both insert and vector) against ladder position for sequence length (i.e. molar weight ratio of the two)
    Rule of 3: Three times as much insert as vector ensures successful ligation results


     

    1. Ligation solution (20 μL) was prepared

    • 10x buffer (2 μL)
    • T4 ligase (2 μL)
    • Vectors and insert (~17 μL), according to calculated ratio
    • make up to a total of 20 μL using water

    2. The ligation mixture was allowed to stand at room temperature for 10-30 minutes..

    3. The ligase-treated vector-insert mixture was mixed with E. Coli (DH5-α)(60 μL), then the standard transformation protocol was followed with shaking for 1 hr; finally LB (200 μL) was added.

    p>

    1. The optimal density (OD) of a portion of the sample (1 mL) was measured in the spectrophotometer using LB as a blank.

    2. If the OD was approximately 0.5 the sample was induced. The sample could be incubated further or diluted if necessary.

    2. IPTG (1 mM) was added.

    1. The E. Coli was spun down in a centrifuge.

    2. The supernatant was discarded and phosphate buffer saline(PBS, 1 mL) was added to resuspend the pellet before the mix was transferred to an Eppendorf tube.

    3. The E. Coli was spun at full speed for 1 min., the supernatant discarded and the pellet was put on ice for storage.

    4. Lysozyme solution (10 mg/mL) was prepared in lysis buffer.

    5. Lysozyme solution (200 μL) was added to each pellet and they were resuspended and vortexed before being incubated in a shaker (37 °C) for 30 min. to 1 h.

    6. The cells were sonicated.

    7. Total sample (10 μL) was removed and 2x dye (10 μL) was added.

    8. \the original tubes were spun for 1 min. at full speed and the supernatant was removed, the mass was resuspended with PBS (200 μL).

    9. Membrane sample (10 μL) was taken from the resuspended solution and 2x dye (10 μL) was added.

    10. The protein samples were denatures on a heating block (95° C or 75°C for membrane protein).

    11. The samples were cooled on ice for 1 min., then loaded on to the protein gel (prepared according to SDS-PAGE standard protocol) and run at 150 V for around 45 min.

    1. Making the membrane transfer sandwiches: 2x A2 at the bottom, then 2x A2.

    2. The membrane, GE Healthcare HyBond ECL films, was cut out using a glass template; the membrane was put onto moist A2 filter papers and pressed flat using a roll.

    3. The gel slides were opened using a wedge; removed the thinner slide and, using the wedge, prized the protein gel from the thicker plate and onto the membrane.

    4. The airbubbles were gently pressed out between the membrane and the gel.

    5. CAT filter paper was added.

    6. The power pack was run for 1hr (40 mA).

    7. If the restained markers have transferred, place the CAT filter papers onto a rocker and add stain. If using Ponceau, the stain can be reused.

    8. Destain was done by rinsing with water.

    21. Milk powder (50 mL 5%) was added and left to rock for 2 hours at room temperature. If leaving overnight, rock in cold room using lower speed.

    9. The blocking milk solution was decanted, and replaced with milk (10 mL 1%) with antibodies (5μl of diluted). This was rocked for 1 h.

    10. The milk-antibody solution was poured off and washed with PBS (0.3%) and Tween, three times at 10 min intervals.

    11. A solution of "Supersignal west Fermto trail" kit (1:1 ratio, 1 mL per membrane).

    12. The membrane was made moist with the visualization solution, covered for 5 min, then the excess liquid was removed.

    13. The membranes were developed in a dark room.


     

    Project-specific procedures

    ω3-Synthesis

    1. The E. Coli was spun down in a centrifuge(800g, 10min).

    2. With the supernatant discarded, PBS (1 mL) was used to resuspend the pellet, then transferred to Eppendorf tubes.

    3. The E. Coli was spun again at full speed for 1 min. and the supernatant discarded.

    4. The pellet was resuspended using PBS (100 μL) and then transferred to glass sample vials.

    5. An organic mixture of chloroform:methanol (1:2(v/v), 375 μL) was added for lysis.

    6. The sample was vortexed for 1 h in the cold room, with chloroform (125 μL) and water (125 μL) added to allow phase separation (lower organic layer contains lipids); and left to stand for at least 10 min.

    7. The sample vials were spun at 1000g at room temperature for 5 min.

    8. The lower organic layer was transfered to a new glass vial avoiding the upper aqueous layer at all costs.

    9. The resultant lower phase lipid extract was then dried under N2, and the fatty acid stored in the fridge.

    9. The samples can be re-dissolved in chloroform:methanol (1:2) and are ready for Electrospray-mass spectrometry analysis for phospholipids and can be further processed for fatty acid derivation and analysis by Gas Chromatography-Mass Spectrometry.

    1. Per cell type, there will be 3 samples:

    • A (negative control)
    • B (no added substrate beyond induced 18:1)
    • C (addition of substrate PC lipid 18:1)


     

    2. After labelling all screw-cap Eppendorfs, add the following assay components to each B and C tube:

    • tricine buffer (20 μL, 40 μM), made up to pH 8 with KOH
    • MgCl2 (20 μL of 10 μM)
    • Ferredoxin (10 μL)
    • NADPH (10 μL, 5μM)
    • Catalase (30 μL)
    • NADPH+ (10 μL)


     

    3. The membrane sample (100 μL), prepare d as in the lipid extraction, was added to the mixture. Lysozyme (500 μL) was added, and resuspended in tricine buffer. The Bradford assay was run to determine protein content.

    4. The sample was vortexed at 4°C for 2 min.

    5. It was put in a 37°C water bath.

    6. Chloroform (250 μL) and water (250 μL) inhibited the assay reaction and allowed for phase separation.

    7. Samples were used to carry out mass spec analysis.

    1. Aliquots of the lipid extract were transferred to 2mL glass vessels and dried under nitrogen.

    2. Base hydrolysis to release fatty acids was done using of concentrated ammonia (500 µL) and 50% propan-1-ol (50%) 1:1, followed by incubation for 5 h at 50 °C.

    3. After cooling, the samples were evaporated to dryness with nitrogen and were dried twice more from methanol : water (1:1) (200 µL) to remove all traces of ammonia.

    4. The protonated fatty acids are then extracted by partitioning between HCl (500 µL, 20 mM) and of ether (500 µL); the aqueous phase was re-extracted with fresh ether (500µL) and the combined ether phases were dried under nitrogen in a glass tube.

    5. The fatty acids were converted to fatty acid methyl esters (FAMEs), by adding diazomethane (3x 20 µL aliquots) to the dried residue, while on ice. After 30min, the samples were allowed to warm to RT and left to evaporate to dryness in a fume hood.

    6. The FAME products were dissolved in dichloromethane (10-20 µL) and analysed (1-2 µL) by GC-MS on a Agilent Technologies (GC-6890N, MS detector-5973) with a ZB-5 column (30M x 25mm x 25mm, Phenomenex), with a temperature program of at 70 °C for 10min followed by a gradient to 220 °C at 5 °C /min and held at 220 °C for a further 15min

    7. Mass spectra were acquired from 50-500 amu. The identity of FAMEs was carried out by comparison of the retention time and fragmentation pattern with a Bacterial FAME standard (Supelco).

    The Bradford protein assay was carried out in accordance with standard procedure as described in BRADFORD, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 7;72:248-54.


     

    Metal-binding peptides

    Primer annealing was carried out to create short peptides.

    10 μL of 200 μM of each primer was put into the PCR machine.
     

    PCR Programme:

  • Step 1 94 °C 1 min
  • Step 2 42 °C 1 min
  • Step 3 72 °C 10 seconds
  • Step 4 To step 1
  • Repeat 5 times.
  • 1. The E. coli was spun down in a centrifuge.

    2. The supernatant was discarded and phosphate buffer saline (PBS, 1 mL) was added to re-suspend the pellet. The mixture was transferred to eppendorf tubes.

    3. The E. coli was spun down at full speed for 1 min. The supernatant was discarded and then put on ice for storage.

    4. Lysozyme solution (10 mg/mL) was prepared in lysis buffer.

    5. Lysozyme solution (200 μL) was added to each pellet and re-suspended. Benzonase nuclease (1 µL) was added and 1 protease inhibitor cocktail pill was disolved in the solution; this was vortexed and incubated in a shaker at 37 °C for 30 min. to 1 h.

    6. The cells were sonicated.

    7. The sonicated cells were spun down at full speed for 1 min in a centrifuge.

    8. The Nickel beads were prepared. Ni-NTA slurry per sample(100 µL). This was spun in centrifuge at full speed, and the supernatant removed . PBS was added (100 µL), the pellet was re-suspended and spun down at full speed for 1 min.

    9. The supernatant was removed; PBS was added (100 µL), the pellet was re-suspended and transferred to eppendorf tubes (100 µL). This was spun down at full speed for 1 minute in a centrifuge,and the supernatant was removed. The supernatant of the sonicated cells was added and the mix was re suspended.

    10. The samples were agitated for 30 min to 3 h in the cold room. The samples were spun down at full speed for 1 minute in centrifuge and the supernatant was removed.

    11. NINTA elution buffer (100 µL) was added and samples were agitated for 10 to 30 min. in the cold room.

    12. The samples were spun down at full speed for 1 min. and 10 µL of supernatant was removed.2x dye was added to the supernatant (10 µL).

    13. The protein samples were denatures on the heating block at 95° C.

    14. The samples were cooled on ice for 1 min., and then loaded to the gel (prepared according to SDS-PAGE standard protocol) and run at 150 V for 45 min.

    1. The E. coli were spun down in a centrifuge.

    2. The supernatant was discarded and phosphate buffer saline (PBS) (1 mL) was added to re-suspend the pellet. The mixture was transferred to eppendorf tubes.

    3. The E. coli in the tubes were spun down at full speed for 1 min. The supernatant was then discarded and stored on ice.

    4. The lysozyme solution (10 mg/mL) was prepared in lysis buffer.

    5. The lysozyme solution (200 μL) was added to each pellet and re-suspended. Benzonase nuclease (1 μL) was added, and one protease inhibitor cocktail pill was dissolved in the mixture. This was then vortexed and incubated in the shaker (37 °C for 30 min to 1 h).

    6. The cells were then sonicated.

    7. The sonicated cells were then spun down at full speed for 1 min in the centrifuge .

    8. GST beads were prepared. GST slurry (100 µL) was added to each sample and spun down at full speed. The supernatant was then removed. PBS (100 µL) was added to each sample, and the mixture re- suspended and spun down at full speed for 1 min.

    9. The supernatant was then removed and PBS (100 µL) added per sample, re-suspended and transferred to eppendorf tubes, 100 µL per sample. The mixture spun down at full speed for 1 min and supernatant was then removed. The supernatant of the sonicated cells was then added.

    10. 10. The samples were agitated for 30 min to 3 h in the cold room. Samples were spun down at full speed for 1 min. Supernatant was removed.

    11. GST beads were washed with PBS, 200 µL per sample. Samples were spun down at full speed and supernatant was removed. Glutathione solution (50 µl, 100 mM) was added . The samples were agitated for 10 to 30 min in the cold room.

    12. The samples were spun at full speed for 1 min. 10 µL of supernatant was taken and dye (10 µL, 2x) was added.

    13. The protein samples were denatured using the heating block at 95° C.

    14. The samples were cooled on ice (1 min) then loaded onto the gel (prepared according to SDS- PAGE standard protocol) and run at 150 V for 45 min.