Team:GeorgiaTech/WeekOne
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- | <p>< | + | <p align="center">8/5/2010</p> |
- | + | <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 |
- | + | <li>Added 350 սL N3, mix immediately, inverted 4-6 times | |
- | + | <li>Centrifuged 10 min, 13k RPM</ol> | |
- | + | <ol><lh><strong>Enzyme digest | |
- | + | </strong> | |
- | + | <li>Poured supernatant into spin column | |
- | Enzyme digest | + | <li>Centrifuged 30-60 seconds and discarded flow-through |
- | + | <li>Washed column with 750 սL PE and centrifuged 30-60 seconds | |
- | + | <li>Discarded flowthrough, centrifuged 1 min | |
- | + | <li>Eluted by placing column in new 1.5 mL tube, added 30 սL EB. Let stand 5 min. | |
- | + | <li>Centrifuged 5 minutes, 5k RPM</li></ol> | |
- | + | ||
- | + | <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). |
- | + | <li>Placed a spin column in a provided 2 mL collection tube. | |
- | + | <li>To bind DNA, applied the sample to the column and centrifuge at 17,900g for 30 – 60 secs. | |
- | + | <li>Discarded flow-through. Placed the column back in the same tube. | |
- | + | <li>To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs. | |
- | + | <li>Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min. | |
- | + | <li>Placed the column in a clean 1.5 mL microcentrifuge tube. | |
- | + | <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. | |
- | + | <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> | |
- | + | ||
- | + | ||
- | + | <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). |
- | + | <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. | |
- | + | <li>After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). | |
- | + | <li>Added 1 gel volume of isopropanol to the sample and mixed. | |
- | + | <li>Placed a QIAquick spin column in a provided 2 mL collection tube. | |
- | + | <li>To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min. | |
- | + | <li>Discarded flow-through and placed QIAquick column back in the same collection tube. | |
- | + | <li>Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min. | |
- | + | <li>To wash, added 0.75 mL of Buffer PE 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). | |
- | + | <li>Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube. | |
- | + | <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. | |
- | + | <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. | |
- | + | ||
- | + |
Latest revision as of 02:47, 28 October 2010
8/5/2010
- Re-suspended pellet in 250 սL PI buffer
- Added 250 սL P2, inverted about 5 times
- Added 350 սL N3, mix immediately, inverted 4-6 times
- Centrifuged 10 min, 13k RPM
- Poured supernatant into spin column
- Centrifuged 30-60 seconds and discarded flow-through
- Washed column with 750 սL PE and centrifuged 30-60 seconds
- Discarded flowthrough, centrifuged 1 min
- Eluted by placing column in new 1.5 mL tube, added 30 սL EB. Let stand 5 min.
- Centrifuged 5 minutes, 5k RPM
- Added 180 mL 1x TBE and 1.8g agarose
- Heated up until agarose was no longer visible
- Let cool (approx. 10 min)
- Added 180 սL Ethidium bromide (1000X)
- 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.
- Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).
- Placed a spin column in a provided 2 mL collection tube.
- To bind DNA, applied the sample to the column and centrifuge at 17,900g for 30 – 60 secs.
- Discarded flow-through. Placed the column back in the same tube.
- To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.
- Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.
- Placed the column in a clean 1.5 mL microcentrifuge tube.
- 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.
- 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.
- Excised DNA fragment from the agarose gel with a clean, sharp scalpel.
- 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).
- 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.
- After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
- Added 1 gel volume of isopropanol to the sample and mixed.
- Placed a QIAquick spin column in a provided 2 mL collection tube.
- To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.
- Discarded flow-through and placed QIAquick column back in the same collection tube.
- Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.
- To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.
- Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
- Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.
- 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.
- 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.