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Team:Westminster/Protocols
From 2012.igem.org
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<h2>PCR Amplification</h2> | <h2>PCR Amplification</h2> | ||
<p>Biolabs Phusion High- Fidelity DNA polymerase was used for all PCR amplifications. The reactions and conditions are given below. </p> <img src="https://static.igem.org/mediawiki/2012/4/4c/Phusion.png" alt="Phusion" title="Reaction Conditions" /> | <p>Biolabs Phusion High- Fidelity DNA polymerase was used for all PCR amplifications. The reactions and conditions are given below. </p> <img src="https://static.igem.org/mediawiki/2012/4/4c/Phusion.png" alt="Phusion" title="Reaction Conditions" /> | ||
| - | <h2> | + | <h2>Conditions:</h2> |
| + | <img src="https://static.igem.org/mediawiki/2012/4/4d/Phusion_conditions.png" alt="Phusion" title="Reaction Conditions" /> | ||
| + | <p>PCR amplification with linkers required an additional 50 M Mg2+ in the mastermix.</p> | ||
| + | <p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p> | ||
<p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p> | <p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p> | ||
<p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p> | <p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p> | ||
Revision as of 18:30, 26 September 2012
AMPLIFICATION OF GENOMIC DNA
Cell Lysis
We used Stratagene kit for lysing human cells and extracting DNA.
• Pellet up to a maximum of 1x 106 cells for 30 sec. in microfuge. If more than 1X 106 cells are used, the reagent volumes will have to be increased accordingly. For adherent cells, a trypsin treatment is generally used prior to this step to free the cells into suspension. Be careful to perform the trypsin step in a timely manner so that5 the cells do not remain in undiluted trypsin for a long period of time.
• Aspirate media and wash cells with 500 l of 1x PBS.
• Pellet cells 30 sec. in microfuge at 14, 000 rpm. • Aspirate PBS and resuspend cells in 500 l of PBS. Repeat spin and aspiration steps. • Resuspend cells in 100 l PBS and 200l sterile water. • Lyse cells by heating to 950C for 10-15 minutes. • Allow the cells to cool briefly by setting at room temperature for 5 min. And add 10 l of 10 mg/ml Proteinase K to each sample. Quick vortex to mix. • Incubate at 550C for one hour. • Inactivate Proteinase K by heating to 950c for 10 minutes. • Spin down condensation. • Store at -200c.PCR Amplification
Biolabs Phusion High- Fidelity DNA polymerase was used for all PCR amplifications. The reactions and conditions are given below.
Conditions:
PCR amplification with linkers required an additional 50 M Mg2+ in the mastermix.
Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.
Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.
Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.
