Minnesota/24 July 2008

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Revision as of 16:24, 24 July 2008

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1. Base Vector Plasmid Maxi Prep Base Vector: Grew another culture of different base vector colonies, so now need to plasmid prep the base vector so the DNA/plasmid will be extracted from the rest of the cell.

2. Spectrophotometry: Excellent results from spec'ing new base vector plasmid. Very high concentration of DNA.

3. Base Vector & Promoter Double Digest: 50uL Reaction Mixture. Digest incubation for 2 hours @ 37C. Heat inactivate enzyme for 15 minutes @ 65C in a water bath. Follow the table below for pre-incubation steps:

Parts	10x Buffer	BSA	H20	DNA from parts	RE 1	RE 2	[DNA] ng/uL
BV	5.0uL	0.5uL	30.8uL	11.7uL	EcoRI, 1.0uL	Pst1, 1.0uL	100.62 ng/uL
TetR Pro. 1	5.0uL	0.5uL	35.5uL	7.0uL	EcoRI, 1.0uL	Pst1, 1.0uL	23.8 ng/uL
Tet R Pro. 2	5.0uL	0.5uL	35.5uL	7.0uL	EcoRI, 1.0uL	Pst1, 1.0uL	23.8 ng/uL
LacI Pro. 1	5.0uL	0.5uL	41.5uL	1.0uL	EcoRI, 1.0uL	Pst1, 1.0uL	168.0 ng/uL
LacI Pro. 2	5.0uL	0.5uL	41.5uL	1.0uL	EcoRI, 1.0uL	Pst1, 1.0uL	168.0 ng/uL
Lac/Lam 1	5.0uL	0.5uL	28.5uL	14.0uL	EcoRI, 1.0uL	Pst1, 1.0uL	9.8 ng/uL
Tet/p22 1	5.0uL	0.5uL	34.0uL	8.5uL	EcoRI, 1.0uL	Pst1, 1.0uL	20.4 ng/uL
Tet/p22 2	5.0uL	0.5uL	34.0uL	8.5uL	EcoRI, 1.0uL	Pst1, 1.0uL	20.4 ng/uL

4. Vector Dephosphorylation: After the digested products' enzyme has been heat inactivated, we dephosphorylated the base vector so the cut ends wouldn't religate. Follow the table below:

Parts	Antarctic Phosphatase	Antarctic Phosphatase Buffer
BV	1.0uL	5.1uL

@@ Line 37: / Line 37: @@
 |-
-|'''4. Vector Dephosphorylation:'''
+|'''4. Vector Dephosphorylation:''' After the digested products' enzyme has been heat inactivated, we dephosphorylated the base vector so the cut ends wouldn't religate. Follow the table below:
+{|border="1" align="left"
+|-
+!| Parts !! Antarctic Phosphatase !! Antarctic Phosphatase Buffer
+|-
+| BV || 1.0uL || 5.1uL
+|}