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| | <div class="sectiontitle"> | | <div class="sectiontitle"> |
| − | <h4>Experiment 1: Testing the binding of specifically designed DNA strands to glass slides</h4> | + | <h4>Experiments</h4> |
| | </div> | | </div> |
| | <p> | | <p> |
| − | | + | <h4>Experiment 1: Testing the binding of specifically designed DNA strands to glass slides</h4> |
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| | <br>Glass slides were prepared (put link to Glass Slide Preparation Protocol) by being cleaned and functionalised (with HCl and GOPTS respectively). | | <br>Glass slides were prepared (put link to Glass Slide Preparation Protocol) by being cleaned and functionalised (with HCl and GOPTS respectively). |
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| − | <h5>Extracting gel</h5>
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| − | <p>After running a gel and identifying the band that contains the DNA that you wish to extract, simply use a scalpel to cut around the band leaving as little excess agarose gel as possible.</p>
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| − |
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| − | <H5>Protocol</H5>
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| − | <br>1. Measure the volume of the DNA sample.
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| − | <br>2. Add 1/10 volume of sodium acetate, pH 5.2, (final concentration of 0.3 M) - These amounts assume that the DNA is in TE only; if DNA is in a solution containing salt, adjust salt accordingly to achieve the correct final concentration.
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| − | <br>3. Mix well.
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| − | <br>4. Add 2 to 2.5 volumes of cold 100% ethanol (calculated after salt addition).
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| − | <br>5. Mix well.
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| − | <br>6. Place on ice or at -20 degrees C for >20 minutes.
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| − | <br>7. Spin a maximum speed in a microfuge 10-15 min.
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| − | <br>8. Carefully decant supernatant.
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| − | <br>9. Add 1 ml 70% ethanol. Mix. Spin briefly. Carefully decant supernatant.
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| − | <br>10. Air dry or briefly vacuum dry pellet.
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| − | <br>11. Resuspend pellet in the appropriate volume of TE or water.
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| − | </p>
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| − |
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| − | <h3> Double restriction digestion for NEB restriction enzymes </h3>
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| − | <h5> Cuts a select piece of DNA from a plasmid </h5>
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| − | <h5> Protocol </h5>
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| − | <p> Set up the reaction as follows:
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| − | <br> - 1ug DNA
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| − | <br> - 5uL 10x digest buffer (use NEB cloner to find which buffer works best with which enzyme)
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| − | <br> - 1uL or 10 units of first enzyme
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| − | <br> - 1uL or 10 units of second enzyme
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| − | <br> - Up to 50uL nuclease-free water
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| − | <br>
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| − | <br> Incubate at 37C for 1 hour. If the enzymes being used are both time save qualified, this can be reduced to 5-15 minutes, but incubating for longer is still recommended.
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| − | <br> Add the reagents into the mix from largest volume to smallest, always finishing with adding the enzymes in last.
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| − | <br> If multiple restriction digests are being set up, a master mix containing everything but the sample DNA can be made with the condition that the concentrations of the different sample DNA are similar or equal.
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| − | </p>
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| − |
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| − | <h3> Bacterial immunofluorescence protocol </h3>
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| − | <h5> For preparing slides to be visualised under fluorescent microscopy </h5>
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| − | <p> 1. For every cell type that needs testing, grow a culture of bacterial cells in 5mL LB
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| − | (+antibiotics) overnight at 37 ˚C.
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| − | <br>2. Next morning, take OD600 of the cultures (OD of 1 for E. coli corresponds to ~10^8 cells/mL),
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| − | and dilute into 2 fresh 5mL LB tubes (+antibiotics) to OD600 of ~0.01. To one of these tubes,
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| − | add IPTG to end concentration of 1mM. Incubate both tubes in a 37 ˚C shaker.
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| − | <br>3. After ~3 hr of incubation, start monitoring OD of the cultures every half hour. We want to fix
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| − | these cells at an OD600 of ~0.5.
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| − | <br>4. As soon as a culture reaches OD600 of ~0.4-0.5, spin down 1mL of the culture in an Eppendorf
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| − | tube at 8000xg (=rcf) for 1 min, and carefully discard the supernatant (be careful so as to
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| − | only remove the supernatant, without disturbing the cells in the pellet).
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| − | <br>5. Re-suspend the pellet in 1mL 1xPBS by pipetting up and down 5 times. Spin down the cells at
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| − | 8000xg (=rcf) for 1 min, and carefully discard the supernatant.
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| − | <br>6. Repeat the PBS wash in Step-5 two more times, but this time only use 0.5mL PBS.
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| − | <br>7. Now, re-suspend the cells in 0.5mL 1xPBS by pipetting.
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| − | <br>8. Mix 500 uL Blocking buffer with the annealed oligo (5.13uL) for each cell type in a separate
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| − | Eppendorf tube, then add this to your cells.
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| − | <br>9. Spin down the cells at 8000xg (=rcf) for 1 min, and carefully discard the supernatant.
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| − | <br>10. Do 1x PBS wash (0.5mL PBS).
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| − | <br>11. Now, fix the cells (in the tube itself) by resuspending in 1xPBS+4%(para)formaldehyde (we used glutaraldehyde but it fulfills the same purpose) (500uL). Incubate at room temperature for 20 min.
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| − | <br>12. Do 1x PBS washes (0.5mL PBS)
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| − | <br>13. Drop 50uL of the resuspension on a coverslip (round coverslips preferred), and incubate at
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| − | 37 ˚C until it is completely dry. Once dry, save the coverslip at RT until all the cultures have
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| − | been processed similarly.
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| − | <br>14. Add a drop of the mounting medium (ProLong Diamond Antifade Reagent, Fisher
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| − | #15372192) on a glass slide and place the coverslip on top of it (bacterial-side-down).
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| − | <br>15. Seal the edges of the cover-slip with nail-polish, and save in the fridge (4˚C), for later
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| − | visualization.
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| − | </p>
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| − | <br>
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| − | <br>
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