Team:GeorgiaTech/WeekOne

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<p><center class="style1">
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<p align="center">8/5/2010</p>
-
 
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<ol><lh><strong>Extracting pCOLD from cells
-
8/5/2010
+
</strong>
-
 
+
<li>Re-suspended pellet in 250 սL PI buffer
-
Extracting pCOLD from cells
+
<li>Added 250 սL P2, inverted about 5 times
-
1. Re-suspended pellet in 250 սL PI buffer
+
<li>Added 350 սL N3, mix immediately, inverted 4-6 times
-
2. Added 250 սL P2, inverted about 5 times
+
<li>Centrifuged 10 min, 13k RPM</ol>
-
3. Added 350 սL N3, mix immediately, inverted 4-6 times
+
<ol><lh><strong>Enzyme digest
-
4. Centrifuged 10 min, 13k RPM
+
</strong>
-
 
+
<li>Poured supernatant into spin column
-
Enzyme digest
+
<li>Centrifuged 30-60 seconds and discarded flow-through
-
5. Poured supernatant into spin column
+
<li>Washed column with 750 սL PE and centrifuged 30-60 seconds
-
6. Centrifuged 30-60 seconds and discarded flow-through
+
<li>Discarded flowthrough, centrifuged 1 min
-
7. Washed column with 750 սL PE and centrifuged 30-60 seconds
+
<li>Eluted by placing column in new 1.5 mL tube, added 30 սL EB. Let stand 5 min.
-
8. Discarded flowthrough, centrifuged 1 min
+
<li>Centrifuged 5 minutes, 5k RPM</li></ol>
-
9. Eluted by placing column in new 1.5 mL tube, added 30 սL EB. Let stand 5 min.
+
 
-
10. Centrifuged 5 minutes, 5k RPM
+
  <center>8/6/2010</center>
-
 
+
 
-
8/6/2010
+
  <ol><lh>Prepared 1% agarose gels:
-
 
+
  <li>Added 180 mL 1x TBE and 1.8g agarose
-
Prepared 1% agarose gels:
+
  <li>Heated up until agarose was no longer visible
-
Added 180 mL 1x TBE and 1.8g agarose
+
  <li>Let cool (approx. 10 min)
-
Heated up until agarose was no longer visible
+
  <li>Added 180 սL Ethidium bromide (1000X)</ol>
-
Let cool (approx. 10 min)
+
 
-
Added 180 սL Ethidium bromide (1000X)
+
  <ol><lh>PCR purifcation of the cell cultures from 8/5/2010
-
 
+
  <li>Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.
-
PCR purifcation of the cell cultures from 8/5/2010
+
  <li>Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).
-
1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.
+
  <li>Placed a spin column in a provided 2 mL collection tube.
-
2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).
+
  <li>To bind DNA, applied the sample to the column and centrifuge at 17,900g for 30 – 60 secs.
-
3. Placed a spin column in a provided 2 mL collection tube.
+
  <li>Discarded flow-through. Placed the column back in the same tube.
-
4. To bind DNA, applied the sample to the column and centrifuge at 17,900g for 30 – 60 secs.
+
  <li>To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.
-
5. Discarded flow-through. Placed the column back in the same tube.
+
  <li>Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.
-
6. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.
+
  <li>Placed the column in a clean 1.5 mL microcentrifuge tube.
-
7. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.
+
  <li>To elute DNA, added 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0 – 8.5) to the center of the membrane and centrifuged the column for 1 min. Alternatively, for increased DNA concentration, added 30  µL elution buffer to the center of the membrane, let the column sit for 1 min, and then centrifuged.
-
8. Placed the column in a clean 1.5 mL microcentrifuge tube.
+
  <li>If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.</ol>
-
9. To elute DNA, added 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0 – 8.5) to the center of the membrane and centrifuged the column for 1 min. Alternatively, for increased DNA concentration, added 30  µL elution buffer to the center of the membrane, let the column sit for 1 min, and then centrifuged.
+
 
-
10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
+
 
-
 
+
  <ol><lh>Gel Extraction Protocol
-
 
+
  <li>Excised DNA fragment from the agarose gel with a clean, sharp scalpel.
-
Gel Extraction Protocol
+
  <li>Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).
-
1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.
+
  <li>Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.
-
2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).
+
  <li>After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
-
3. Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.
+
  <li>Added 1 gel volume of isopropanol to the sample and mixed.
-
4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
+
  <li>Placed a QIAquick spin column in a provided 2 mL collection tube.
-
5. Added 1 gel volume of isopropanol to the sample and mixed.
+
  <li>To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.
-
6. Placed a QIAquick spin column in a provided 2 mL collection tube.
+
  <li>Discarded flow-through and placed QIAquick column back in the same collection tube.
-
7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.
+
  <li>Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.
-
8. Discarded flow-through and placed QIAquick column back in the same collection tube.
+
  <li>To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.
-
9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.
+
  <li>Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
-
10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.
+
  <li>Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.
-
11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
+
  <li>To elute DNA, added 50  μL of Buffer EB (1 mM Tris-Cl, pH 8.5) or water (pH 7.0 – 8.5) to the center of the QIAquick membrane and centrifuged the column for 1 min. Alternatively, for increased DNA concentration, added 30  μL elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuged for 1 min.
-
12. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.
+
  <li>If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
-
13. To elute DNA, added 50  μL of Buffer EB (1 mM Tris-Cl, pH 8.5) or water (pH 7.0 – 8.5) to the center of the QIAquick membrane and centrifuged the column for 1 min. Alternatively, for increased DNA concentration, added 30  μL elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuged for 1 min.
+
-
14. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
+

Latest revision as of 02:47, 28 October 2010

Georgia Institute of Technology iGEM Team 2010 Homepage

mainbanner menubar Home Project Notebook Modeling Parts Ethics & Safety Team Sponsors Team Contacts

8/5/2010

    Extracting pCOLD from cells
  1. Re-suspended pellet in 250 սL PI buffer
  2. Added 250 սL P2, inverted about 5 times
  3. Added 350 սL N3, mix immediately, inverted 4-6 times
  4. Centrifuged 10 min, 13k RPM
    Enzyme digest
  1. Poured supernatant into spin column
  2. Centrifuged 30-60 seconds and discarded flow-through
  3. Washed column with 750 սL PE and centrifuged 30-60 seconds
  4. Discarded flowthrough, centrifuged 1 min
  5. Eluted by placing column in new 1.5 mL tube, added 30 սL EB. Let stand 5 min.
  6. Centrifuged 5 minutes, 5k RPM
8/6/2010
    Prepared 1% agarose gels:
  1. Added 180 mL 1x TBE and 1.8g agarose
  2. Heated up until agarose was no longer visible
  3. Let cool (approx. 10 min)
  4. Added 180 սL Ethidium bromide (1000X)
    PCR purifcation of the cell cultures from 8/5/2010
  1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.
  2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).
  3. Placed a spin column in a provided 2 mL collection tube.
  4. To bind DNA, applied the sample to the column and centrifuge at 17,900g for 30 – 60 secs.
  5. Discarded flow-through. Placed the column back in the same tube.
  6. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.
  7. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.
  8. Placed the column in a clean 1.5 mL microcentrifuge tube.
  9. To elute DNA, added 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0 – 8.5) to the center of the membrane and centrifuged the column for 1 min. Alternatively, for increased DNA concentration, added 30 µL elution buffer to the center of the membrane, let the column sit for 1 min, and then centrifuged.
  10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
    Gel Extraction Protocol
  1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.
  2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).
  3. Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.
  4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
  5. Added 1 gel volume of isopropanol to the sample and mixed.
  6. Placed a QIAquick spin column in a provided 2 mL collection tube.
  7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.
  8. Discarded flow-through and placed QIAquick column back in the same collection tube.
  9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.
  10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.
  11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
  12. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.
  13. To elute DNA, added 50 μL of Buffer EB (1 mM Tris-Cl, pH 8.5) or water (pH 7.0 – 8.5) to the center of the QIAquick membrane and centrifuged the column for 1 min. Alternatively, for increased DNA concentration, added 30 μL elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuged for 1 min.
  14. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.