Team:UCL London/Glycerol Stock

(Difference between revisions)

Revision as of 13:53, 30 September 2010

UCL IGEM 2010

Materials

Glycerol

Transformed E.coli cells.

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.

@@ Line 20: / Line 20: @@
 .	Rest of the E.coli suspension are stored in the fridge.
-'''Miniprep Material'''
-Miniprep Kit
-===Method===
-.	Label Set 1 eppendorfs; Label Spin column tubes; Label Set 2 eppendorfs.
-.	Add ~1.5ml E.coli suspension in Set 1 eppendorfs; and then centrifuge for 3min at 8000rpm & discard all the supernatant.
-.	Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
-.	Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
-.	Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
-.	Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
-.	Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
-.	Centrifuge for 30–60 s. Discard the flow-through.
-.	Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard
-the flow-through.
-.	Recommended: Wash the QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. Discard
-the flow-through.
-.	Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
-.	Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM
-Tris•Cl, pH 8.5) # Store at -20°C.
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