Team:CIDEB-UANL Mexico/Test/Protocol
From 2012hs.igem.org
(Difference between revisions)
Line 538: | Line 538: | ||
<p><li>1.Take 1ml of mQ water and place it inside a 1.5mL centrifuge tube.</li> | <p><li>1.Take 1ml of mQ water and place it inside a 1.5mL centrifuge tube.</li> | ||
- | 2.Add 1 μL of plasmidic DNA samples of E.coli (Dilution 1:1,000).</li> | + | <li>2.Add 1 μL of plasmidic DNA samples of E.coli (Dilution 1:1,000).</li> |
- | 3.Calibrate the spectrophotometer with a cuvette containing 1mL of distillated water.</li> | + | <li>3.Calibrate the spectrophotometer with a cuvette containing 1mL of distillated water.</li> |
- | 4.Transfer the sample of the centrifuge tube to a cell of the spectrophotometer by using a micropipette.</li> | + | <li>4.Transfer the sample of the centrifuge tube to a cell of the spectrophotometer by using a micropipette.</li> |
- | 5.Place the cell in the spectrophotometer.</li> | + | <li>5.Place the cell in the spectrophotometer.</li> |
- | 6.Select the DNA or RNA option in the machine.</li> | + | <li>6.Select the DNA or RNA option in the machine.</li> |
- | 7.Select the DNA option in the spectrophotometer.</li> | + | <li>7.Select the DNA option in the spectrophotometer.</li> |
- | 8.Read the absorbance of the sample with the spectrophotometer.</li> | + | <li>8.Read the absorbance of the sample with the spectrophotometer.</li> |
- | 9.Annotate the reading made at 260, 280 and 320 nm, the relation 260/280 and the concentration given by the spectrophotometer.</li> | + | <li>9.Annotate the reading made at 260, 280 and 320 nm, the relation 260/280 and the concentration given by the spectrophotometer.</li> |
- | 10.Remove the cell from the spectrophotometer and throw away its containing into a biological wastes container.</li> | + | <li>10.Remove the cell from the spectrophotometer and throw away its containing into a biological wastes container.</li> |
- | 11.Wash the cell, first with distillated water and then with ethanol at 100% and put it in a chemical wastes container.</li> | + | <li>11.Wash the cell, first with distillated water and then with ethanol at 100% and put it in a chemical wastes container.</li> |
<li>12.Let the cell dry so it can be reused. (It’s recommended to use the same used solution).</li></p> | <li>12.Let the cell dry so it can be reused. (It’s recommended to use the same used solution).</li></p> | ||
<div class="br"></div><div class="br"></div> | <div class="br"></div><div class="br"></div> | ||
Line 555: | Line 555: | ||
<h2>Plasmidic DNA characterization</h2> | <h2>Plasmidic DNA characterization</h2> | ||
- | <p><li>1.Prepare the mix.</li> | + | <p><li>1.Prepare the digestion mix with the restriction enzymes needed ( Reaction order: mQ water enzyme > Buffer > Enzyme buffer > DNA to get a final volume of 10 μL).</li> |
<li>2.Distribute the mix in equal parts.</li> | <li>2.Distribute the mix in equal parts.</li> | ||
- | <li>3.Add the sample of DNA to the reactions and give a | + | <li>3.Add the sample of DNA to the reactions and give a gently vortex.</li> |
- | <li>4.Incubate the reactions at 37°C in the incubator from 1 to 12 hours. | + | <li>4.Incubate the reactions at 37°C in the incubator from 1 to 12 hours.</p> |
- | 5.Run the agarose to check the result.</p> | + | <li>5.Run the agarose to check the result.</p> |
<div class="br"></div><div class="br"></div> | <div class="br"></div><div class="br"></div> | ||
<div class="br"></div><div class="br"></div> | <div class="br"></div><div class="br"></div> | ||
Line 566: | Line 566: | ||
<h2>BioBrick Pieces Assembly</h2> | <h2>BioBrick Pieces Assembly</h2> | ||
- | <p><li>1.Prepare the mix. (Reaction order: mQ water > Buffer > Enzyme).</li> | + | <p><li>1.Prepare the mix. (Reaction order: mQ water > Buffer > Enzyme). Using the NEB enzymes.</li> |
<li>2.Distribute the mix in equal parts.</li> | <li>2.Distribute the mix in equal parts.</li> | ||
- | <li>3.Add the sample of DNA to the reactions and give a | + | <li>3.Add the sample of DNA to the reactions and give a gently vortex.</li> |
<li>4.Incubate the reactions at 37°C in the incubator from 1 to 12 hours.</li> | <li>4.Incubate the reactions at 37°C in the incubator from 1 to 12 hours.</li> | ||
<li>5.Run 10 μL in agarose gel.</li> | <li>5.Run 10 μL in agarose gel.</li> | ||
Line 576: | Line 576: | ||
<p><li>1.Take in account the concentration of every sample that now contain the fragments which will used bind.</li> | <p><li>1.Take in account the concentration of every sample that now contain the fragments which will used bind.</li> | ||
- | <li>2.Use the Ligation Calculator to obtain the quantities to separate the ligation MIX | + | <li>2.Use the Ligation Calculator to obtain the quantities to separate the ligation MIX with a final volume of 20 μL.</li> |
- | <li>3.Prepare the mix using the quantities given by the calculator in the following order: Agua mQ >Ligation Buffer>Vector/Fragment.</li> | + | <li>3.Prepare the mix using the quantities given by the calculator in the following order: Agua mQ >Ligation Buffer>Vector/Fragment ratio.</li> |
- | <li>4.Distribute the ligation mix if necessary and add the ligase T4 | + | <li>4.Distribute the ligation mix if necessary and add the ligase T4 (NEB ligase)</li> |
- | <li | + | <li>5.Incubate the reactions at 25°C (room temperature) for an 1 hour or all night long.</li></p> |
</div> | </div> | ||
Revision as of 22:06, 16 June 2012
Lab Protocols
Use of plates with BioBrick lyophilazed