<li>5µl gel red stain

                 <li>3. Prepare the samples by adding the appropriate amount of loading dye (e.g. 5µl dye per 20µl sample)

               Culture Protocol

                 <li>10µl Specific Antibiotic (Chloramphenicol, Ampicillin or Kanamycin)

                 <li>1. Pour 10ml of LB into a 15ml falcon tube.  

                 <li>2. Pipette 10ml of antibiotic into the broth.

                 <li>3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to falcon tube.

             Gel Purification Protocol

                 <li>3.  Add 300µl of Buffer QG to each 100mg of gel.

                 <li>4. Incubate at 50°C for 10 min or until the gel has completely dissolved. Mix by vortexing the tube every 2 min during the incubation.

                 <li>9. Place column back in same collection tube. Add 500µl of Buffer QG to the column and centrifuge for 1 min to remove all traces of agarose.

                 <li>10. Wash column by adding 750µl buffer PE. Let it stand for 2-5 min and then centrifuge for 1 min.  

                 <li>11. Discard the flow-through. Centrifuge for 1 min to remove the residual buffer PE.  

                 <li>12. Then place the column in a clean, labelled 1.5ml microcentrifuge tube.

                 <li>13. To elute the DNA, add 25µl of Buffer EB to the centre of the column membrane, let it stand for 1 min and then centrifuge for 1 min.  

                 <li>14. Using a pipette, transfer the flow-through back into the centre of the column. Let it stand for 1 min and then centrifuge for 1 min. The DNA will now be in the flow-through.

             Glycerol Stock Protocol

                 <li>1. Add the 50% glycerol to a sterile tube.

             Heat Shock Transformation Protocol

                 <li>2.5µl DNA suspension

                 <li>Petri dish with LB agar and specific antibiotic (chloramphenicol, ampicillin or kanamycin)

                 <li>1. Thaw the competent cells on ice.

                 <li>4. Incubate tube on ice for 30 min.

                 <li>6. Place sample back on ice for 3 min.

                 <li>7. Add 250µl of LB to each tube.

                 <li>8. Incubate the cells at 37°C for 1 hour.

                 <li>9. Prepare two plates for each transformation, one with 200µl of cells and another with 100µl of cells.  

             Miniprep Protocol

                 <li>250µl Buffer P1 (Suspension Buffer)

                 <li>250µl Buffer P2 (Lysis Buffer)

                 <li>1. Centrifuge 10ml cell culture at 4,500 x g for 5 mins and pour off the supernatant. Centrifuge at 4,500 x g for 2 min and remove the supernatant by pipetting.

                 <li>2. Resuspend pelleted cells in 250 µl Buffer P1 and transfer the solution to a labelled microcentrifuge tube.  

                 <li>3. Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.  

                 <li>5. Centrifuge for 10 min at 13,000 rpm in a microcentrifuge. A compact white pellet will form.  

                 <li>10. Place the QIAprep column in a clean, labelled microcentrifuge tube. To double elute DNA, add 50 µl Buffer EB to the centre of each QIAprep Spin Column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm. Re-apply flow-through to the centre of the QIAprep Spin Column, let stand for 1 min and centrifuge for 1 min at 13,000 rpm.

             Nanodrop Protocol

             PCR Purification Protocol

                 <li>2. The pH indicator in buffer PB will cause the mixture to turn yellow. If the mixture is orange or violet, add 10µl of 3M sodium acetate and mix until a yellow colour appears.

                 <li>3. Place a QIAquick spin column into a 2ml collection tube.

                 <li>8. Place the column in a clean 1.5ml microcentrifuge tube.

                 <li>9. Elute the DNA by adding 25µl buffer EB to the centre of the membrane in the column. Let it stand for 1 min then centrifuge for 1 min.  

                 <li>10. Using a pipette, transfer the flow-through back into the centre of the column. Let it stand for 1 min and then centrifuge for 1 min.  

             Resuspending gBlocks Protocol

                 <li>100µl buffer TE

                 <li>2. Add 100µl buffer TE to the tube.

             Sequencing Protocol

                 <li>0.32µl 10µM Forward and reverse primers

                 <li>1. Design primers specific to the region of DNA to be sequenced.

                <li>2. Prepare the DNA for sequencing by adding primers and water to a final volume of 6µl.

                 <li>3. Prepare two samples for each DNA sequence, one containing the forward primer, and another separate sample containing the reverse primer.

                 <li>4. Send the samples to be Sanger sequenced by Edinburgh genomics using an ABI 3730 DNA analyser.

                 <li>5. Edinburgh genomics will send an email with the sequence data.