From 2008.igem.org

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 {{:Team:Newcastle University/Header}}
 {{:Team:Newcastle University/Template:UnderTheWetLab|page-title=[[Team:Newcastle University/Protocols|Protocols]]}}
+{{:Team:Newcastle University/WetlabSideBar}}
+<div id="maincol">
+==Protocols==
 [[Agarose Gel Electrophoresis]]
+[[Making Agar Plates]]
 [[Isolating Plasmid from Cells (Miniprep)]]
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 [[Restricting Plasmids (Double Restriction)]]
-[[Ligation]]
+[[Purifying DNA from an enzymatic reaction]]
-[[Transformation]]
-[[Making Frozen Glycerol Cell Stocks]]
-== Ligation ==
-To ligate our 2.2kb ncl08 fragment from pUC57-ncl08 into our 2 vector plasmids (pJWV021 and pGFP-rrnB), we followed the protocol below.  Prior to ligation we did not isolate the 2.2kb fragment from the other resulting digest product (a 2.7kb fragment) because we will select for cells with the correct insert once we have transformed and plated the cells.
-* 2.5μl of 10 x ligase buffer
+[[Cutting a Specific Band from Agarose Gel]]
-* 6μl vector plasmid cut with relevant enzyme(s)
-* 15μl of digested plasmid which contains the fragment to be inserted, digested with the same enzyme(s)as the vector plasmid
-* 1.5μl of T4 ligase
-= 25μl total volume
+[[Purifying DNA from Gel Slices]]
-'''Control:'''
+[[Ligating DNA]]
-* 2.5μl of 10 x ligase buffer
-* 6μl of vector plasmid cut with relevant enzyme(s)
-* 15μl of MilliQ H2O
-* 1.5μl of T4 ligase
-= 25μl total volume
+[[Transforming into DH5α or TOP10 E. coli]]
+[[Making Frozen Glycerol Cell Stocks From Overnight Cultures]]
-== Transformation after ligation ==
+[[Making Overnight Cultures from Frozen Glycerol Cell Stocks]]
-* Add 2μl plasmid to 50μl of competent cells.
+[[Making Overnight Cultures from Agar Colonies]]
-* Incubate for 30 minutes on ice.
-* Heat shock for 2 minutes at 42°C.
-* Add 250μl LB.
-* Incubate for 120 minutes at 37°C whilst shaking or rotating.
+[[Transforming into Bacillus subtillis]]
-===Making Frozen Glycerol Cell Stocks===
+[[Inducing Bacillus subtilis with Subtilin]]
-* Pippette 2μl overnight culture into an Eppendorf tube.
+[[Preparing Bacillus subtilis for Microscopy]]
-* Add 0.5ml glycerol and vortex briefly to mix.
+</div>
-* Prepare a freeze box by decanting a small amount of liquid nitrogen into the box. (N.B: wear protective gloves and face mask.)
-* Place the tube in the box and keep at -80°C.