document.getElementById("tab1").innerHTML = '<p><h2>Standard iGEM protocols</h2><p>Here we have gathered protocols that most iGEM teams use during their everyday lab work. For example making competent cells and the cloning cycle. Most of our standard protocols originates from a book written with help from previous iGEM Uppsala teams and can therefore not be published here. However we advise you to purchase the book since it is very educational and includes several improved protocols.<br><br>Following protocols were taken from “Synthetic Biology, a lab manual”, chapter 6. Protocols.</p><br><br><ul><li>1. 0.9% NaCl</li><li>2. 50% Glycerol</li><li>3. 1M CaCl2</li><li>4. 10x TBE Buffer</li><li>5. SOB Medium</li><li>6. LB Medium</li><li>7. LB Agar Plates and Addition of Antibiotics</li><li>8. Overnight Cultures with antibiotics, and glycerol stock</li><li>9. Agarose gel electrophoresis</li><li>10. Preparation of competent E.Coli cells using CaCl2</li><li>11. Transformation of CaCl2 competent E.coli cells</li><li>12. Bacterial re-streak techniques</li><li>13. Inverse PCR mutagenesis</li><li>14. Digestion with DpnI</li><li>15. Ligation</li><li>16. Colony PCR</li></ul><br><br><p>BioBrick 3A assembly according to NEB protocol, performed with NEB reagents.PCR according to manufacturers protocol. We used NEBs polymerase Q5 for important PCR-reactions and Thermofishers REDTAQ for screening re-streaked colonies.<br><br>All codon optimisation was done for general Escheria coli using a web tool:<br><a href="http://eu.idtdna.com/CodonOpt">http://eu.idtdna.com/CodonOpt</a></p><h3>Sonication</h3><br><p>Day 1 <br>Grow a culture of the desired constructs in LB 16-20 h (37 degrees Celsius) over night. (Don’t forget to also prepare negative controls, one culture without the construct being assessed.</p><br><br>Day 2<br>Centrifuge the samples for 15 min at 3000 g. Discard the supernatant and resuspend the pellet in 500 µl of 20 mM TRIS buffer. Make sure too much time does not pass between centrifugation and discarding of the supernatant.</p><table id="partsT"><tr><th>TRIS Buffer pH 8</th></tr><tr><td>20 mM TRIS</td></tr><tr><td>50 mM NaCl</td></tr></table><ul><li>Add lysozyme, the final concentration should be 0,02 mg/ml</li><li>Transfer the samples to 2 mL eppendorf tubes. Lyse the cells by sonication.</li><li>Centrifuge the tubes for 15 min at maximum speed. (transfer to smaller tubes to be able to centrifuge at a higher speed if needed)</li><li>Transfer the supernatant to new tubes</li></ul><h3>IMAC colon</h3><p><b>Charging the column with metal ions</b></p><ul><li>1. Prepare a 0.1 M stock solution of the desired metal ion (Cu2+, Zn2+, Ni2+, Co2+, Fe3+, etc.) in distilled water.</li> <li>2. Wash the column with 5 column volumes (CV) of distilled water.</li><li>3. Apply1 CV of 0.1M metal ion solution to the column.</li><li>4. Wash the column with at least 5 CV of distilled water to remove excess metal ions. Note that the use of buffer instead of water immediately before and after the application of metal ion solution may cause precipitation.</li><li>5. Wash with 5 CV of the buffer that has been chosen for the protein elution, e.g. imidazole elution buffer for competitive elution. Do NOT use EDTA/EGTA regeneration buffer at this stage!</li><li>6. Equilibrate with 10 CV of binding buffer (20 mM IMAC buffer).The column is now ready for use.</li></ul><br><br><ul><li>1. Save a small aliquot of the filtered lysate for further controls. Gently mix your filtered lysate with 1/3 volume of 4x IMAC buffer. Load the lysate on the IMAC gel at the rate of 1 ml/min. Collect the throughput for safety in one batch, label and store.</li> <li>2. Wash the column with 15 CV of 20 mM IMAC buffer, or overnight. Collect again for safety, label and store.</li><li>3. Wash the column with 15 CV of 100 mM IMAC buffer. Collect also this time, label and store.</li><li>4. Elute with 300 mM IMAC buffer and collect the first 30 mL eluent into 1.5 mL Eppendorf tubes.</li><li>5. Identify fractions containing StEH1 by means of the activity assay.</li></ul><h3>SDS-page</h3><p>Make sure you have these things ready before starting:</p><ul><li>4x Seprataion buffer</li><li>8x Stacking buffer</li><li>8x Running buffer</li><li>30% Acrylamidsol</li><li>10% APS</li><li>TEMED</li><li>The samples ready</li><li>Destainer</li><li>1 M DTT</li></ul><b>Day 1</b><p>Cast two 10 % acrylamide gels<br>Caution! Acrylamide is poisonous, always wear gloves, lab coat and safety glasses!</p><i><b>Separation gel</b> (the protocol covers the making of <u>two gels</u>)</i><table id="partsT" style="width: 100%"><tr><th></th><th>12%</th><th><b>10%</b></th><th>8%</th><th>7.5%</th></tr><tr><td>4xSeparationbuffer</td><td>2.5 ml</td><td><b>2.5 ml</b></td><td>2.5 ml</td><td>2.5 ml</td></tr><tr><td>30% 30:08 Acrylamid sol.</td><td>4 ml</td><td><b>3.33 ml</b></td><td>2.7 ml</td><td>2.5 ml</td></tr><tr><td>H₂O</td><td>3.35 ml</td><td><b>4.02 ml</b></td><td>4.65 ml</td><td>4.85 ml</td></tr><tr><td>10% APS</td><td>50 µl*</td><td><b>100 µl</b></td><td>50 µl*</td><td>50 µl*</td></tr><tr><td>TEMED</td><td>5 µl*</td><td><b>10 µl</b></td><td>5 µl*</td><td>5 µl*</td></tr></table><i>* Alterations might be needed.</i><br><br><i><b>Stacking gel</b> (the protocol is enough for <u>two gels</u>)</i><table id="partsT" style="width: 100%;"><tr><th></th><th>4%</th><th><b>5%</b></th></tr><tr><td>8 x Stacking Buffer</td><td>625 µl</td><td><b>625 µl</b></td></tr><tr><td>30% 30:08 Acrylamid sol.</td><td>650 µl</td><td><b>833 µl</b></td></tr><tr><td>H₂O</td><td>3.7 ml</td><td><b>3.5 ml</b></td></tr><tr><td>10% APS</td><td>50 µl*</td><td><b>100 µl</b></td></tr><tr><td>TEMED</td><td>5 µl*</td><td><b>10 µl</b></td></tr></table><i>* Alterations might be needed</i>';