Team:UCL London/Glycerol Stock

From 2010.igem.org

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==Glycerol Stock==
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=='''Glycerol Stock'''==
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[[Image:UCL-Plate2.JPG |300px|right]]
'''Materials'''
'''Materials'''
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Glycerol
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  Glycerol
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Transformed E.coli cells.
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  Transformed E.coli cells.
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===Method===
 
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1. Add 80µL glycerol into each labelled eppendorf.
 
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2. Add 300µL E.coli suspension into the glycerol.
 
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3. Vortex & store at -20°C.
 
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4. Rest of the E.coli suspension are stored in the fridge.
 
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'''Miniprep Material'''
 
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Miniprep Kit
 
===Method===
===Method===
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1. Label Set 1 eppendorfs; Label Spin column tubes; Label Set 2 eppendorfs.
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1. Add 80µL glycerol into each labelled eppendorf.
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2. Add ~1.5ml E.coli suspension in Set 1 eppendorfs; and then centrifuge for 3min at 8000rpm & discard all the supernatant.
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2. Add 300µL E.coli suspension into the glycerol.
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3. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
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3. Vortex & store at -20°C.
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4. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
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4. Rest of the E.coli suspension are stored in the fridge.
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5. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
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6. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
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7. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
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8. Centrifuge for 30–60 s. Discard the flow-through.
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9. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard
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the flow-through.
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10. Recommended: Wash the QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. Discard
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the flow-through.
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11. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
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12. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM
 
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Tris•Cl, pH 8.5) # Store at -20°C.
 
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Latest revision as of 20:48, 24 October 2010

UCL IGEM 2010

RETURN TO IGEM 2010

Glycerol Stock

UCL-Plate2.JPG

Materials 

  Glycerol

  Transformed E.coli cells.




Method

1. Add 80µL glycerol into each labelled eppendorf.

2. Add 300µL E.coli suspension into the glycerol.

3. Vortex & store at -20°C.

4. Rest of the E.coli suspension are stored in the fridge.


Retrieved from "http://2010.igem.org/Team:UCL_London/Glycerol_Stock"