Team:Queens Canada/Notebook/Protocols/Rehydration

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<classred1> <b> Rehydration </b></classred1>
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<classred1> <b> rehydration </b></classred1>
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<h3red> Title </h3red>
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<h3red> Rehydration- iGEM Standard Parts</h3red> <p>
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<regulartext> <b> Storage and Labelling </b> </regulartext> <br>
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<regulartext> - Rehydrate primers just before they are needed for PCR </regulartext> <br>
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<regulartext> - Store primers (both concentrations) in the -20C freezer </regulartext> <p>
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<regulartext> <b> Materials </b> </regulartext> <br>
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<regulartext> - Dry primers </regulartext> <br>
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<regulartext> - ddH2O </regulartext> <p>
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<regulartext> <b> Procedure </b> </regulartext> <br>
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<regulartext> 1. Ensure that you know what the primer that you are rehydrating is for so that you can label it properly.  </regulartext> <br>
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<regulartext> 2. Using sterile technique, add the correct volume of ddH2O to the dry primers such that they make a 100μM solution (consult the shipping invoice to see what the volume of water should be). If the volume specified by the invoice is over 900μL, add half the specified volume (to make a 200μM solution). Label the top of the tube with the concentration of the primer.    </regulartext> <br>
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<regulartext> 3. Briefly vortex the tube to ensure that all the DNA has dissolved (make sure there are no white flecks remaining in the bottom of the tube).  </regulartext> <br>
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<regulartext> 4. Let the tube sit for 1-3 minutes to ensure that the primers have completely dissolved. It is convenient to label your tubs during this time. <br>
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5. Briefly vortex again.  <br>
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6. Aliquot out the correct volume of the concentrated (100 or 200μM) primer solution into an eppendorf tube and dilute it to 100μL solution at the concentration of 10μM (if your concentrated primer solution is 100μM, use 10μL or the concentrated primer and 90μL of water to make your 10μM primer solution).
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<h3red> Rehydration- Primers</h3red> <p>
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<regulartext> <b> Storage and Labelling </b> </regulartext> <br>
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<regulartext> - Use 2μL of the DNA solution to transform bacteria (in order to amplify the part).  Save the remainder of the aqueous DNA in the -20°C freezer. </regulartext> <br>
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<regulartext> - Product should be labelled as "DNA (AQ)," using the standard labeling technique, as outlined in the front of your lab book and on the Google Doc. </regulartext> <p>
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<regulartext> <b> Materials </b> </regulartext> <br>
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<regulartext> - Kit plate, from parts distribution </regulartext> <br>
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<regulartext> - ddH2O </regulartext> <p>
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<regulartext> <b> Procedure </b> </regulartext> <br>
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<regulartext> 1. Ensure that you know the correct year, plate and well for your part.  TRIPLE CHECK THIS. Wells are numbered and lettered as shown to the right. <br>
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2. Using sterile technique, load 10μL of diH2O into a p20 pipette.<br>
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3. Pierce the foil of the correct well on the distribution plate, firmly but carefully lower the pipette tip into the very bottom of the well.<br>
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4. Mix the DNA with the water by slowly pumping the water in and out of the pipette tip.  You should see the water turn blue if you've done this correctly.<br>
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5. Leaving all of the water in the well, stretch parafilm over the plate and then replace its cover.  <br>
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6. Write, "DO NOT TOUCH, KEEP LEVEL" on a piece of tape and affix the tape to the lid of the plate.<br>
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7. Ensuring that you keep the plate level, put it into a 4°C fridge, and leave it there for one hour to allow all of the DNA to dissolve.<br>
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8. Transfer the entire DNA solution from the well into an eppendorf tube.<br>
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Latest revision as of 20:32, 28 September 2011

Queen's
rehydration
Rehydration- iGEM Standard Parts

Storage and Labelling
- Rehydrate primers just before they are needed for PCR
- Store primers (both concentrations) in the -20C freezer

Materials
- Dry primers
- ddH2O

Procedure
1. Ensure that you know what the primer that you are rehydrating is for so that you can label it properly.
2. Using sterile technique, add the correct volume of ddH2O to the dry primers such that they make a 100μM solution (consult the shipping invoice to see what the volume of water should be). If the volume specified by the invoice is over 900μL, add half the specified volume (to make a 200μM solution). Label the top of the tube with the concentration of the primer.
3. Briefly vortex the tube to ensure that all the DNA has dissolved (make sure there are no white flecks remaining in the bottom of the tube).
4. Let the tube sit for 1-3 minutes to ensure that the primers have completely dissolved. It is convenient to label your tubs during this time.
5. Briefly vortex again.
6. Aliquot out the correct volume of the concentrated (100 or 200μM) primer solution into an eppendorf tube and dilute it to 100μL solution at the concentration of 10μM (if your concentrated primer solution is 100μM, use 10μL or the concentrated primer and 90μL of water to make your 10μM primer solution).

Rehydration- Primers

Storage and Labelling
- Use 2μL of the DNA solution to transform bacteria (in order to amplify the part). Save the remainder of the aqueous DNA in the -20°C freezer.
- Product should be labelled as "DNA (AQ)," using the standard labeling technique, as outlined in the front of your lab book and on the Google Doc.

Materials
- Kit plate, from parts distribution
- ddH2O

Procedure
1. Ensure that you know the correct year, plate and well for your part. TRIPLE CHECK THIS. Wells are numbered and lettered as shown to the right.
2. Using sterile technique, load 10μL of diH2O into a p20 pipette.
3. Pierce the foil of the correct well on the distribution plate, firmly but carefully lower the pipette tip into the very bottom of the well.
4. Mix the DNA with the water by slowly pumping the water in and out of the pipette tip. You should see the water turn blue if you've done this correctly.
5. Leaving all of the water in the well, stretch parafilm over the plate and then replace its cover.
6. Write, "DO NOT TOUCH, KEEP LEVEL" on a piece of tape and affix the tape to the lid of the plate.
7. Ensuring that you keep the plate level, put it into a 4°C fridge, and leave it there for one hour to allow all of the DNA to dissolve.
8. Transfer the entire DNA solution from the well into an eppendorf tube.

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