Minnesota/17 July 2008

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Revision as of 22:25, 17 July 2008

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1. Plasmid prep dual promoters: (1) Tet & P22mnt, (2) LacI & LAMBDAcI.

2. Model: Figure out why have such a low GFP output when run model.

3. Sequence L5, L6-L9

4. Re-transform RFP, TetR promoter, terminator, and MCherry because no cell growth on plates. Place in 2mL LB cultures, allow growth in incubator @37C for 2 hours. Plate samples on Ampicillin resistant plates. Allow growth O/N.

5. Discuss problems with sequences

6. Double digest of all ligations: from previous days. Do a double digest to cut the components linearly, which will then be able to run through a gel to check if correct DNA is present. Follow the table below:

Parts	10x Buffer	BSA	H20	DNA	RE 1	RE 2
L2a, LAMBDAcI:Term	5.0uL	0.5uL	32.5uL	10.0uL	EcoRI	Xba1
L2b, LAMBDAcI:Term	5.0uL	0.5uL	32.5uL	10.0uL	EcoRI	Xba1
L3i, Pro:LAMBDAcI	5.0uL	0.5uL	32.5uL	10.0uL	Pst1	Spe1
L3j, Pro:LAMBDAcI	5.0uL	0.5uL	32.5uL	10.0uL	Pst1	Spe1
L4b, LAMBDAcI:Term	5.0uL	0.5uL	40.5uL	2.0uL	EcoRI	Xba1
L4c, LAMBDAcI:Term	5.0uL	0.5uL	39.5uL	3.0uL	EcoRI	Xba1
L4d, LAMBDAcI:Term	5.0uL	0.5uL	39.5uL	3.0uL	EcoRI	Xba1
L4e, LAMBDAcI:Term	5.0uL	0.5uL	39.5uL	3.0uL	EcoRI	Xba1
L4g, LAMBDAcI:Term	5.0uL	0.5uL	39.5uL	3.0uL	EcoRI	Xba1
L4h, LAMBDAcI:Term	5.0uL	0.5uL	39.5uL	3.0uL	EcoRI	Xba1
L4i, LAMBDAcI:Term	5.0uL	0.5uL	37.5uL	5.0uL	EcoRI	Xba1
L4j, LAMBDAcI:Term	5.0uL	0.5uL	32.5uL	10.0uL	EcoRI	Xba1
L5c, GFP:Term	5.0uL	0.5uL	40.5uL	2.0uL	EcorI	Xba1
L6a, Pro:LAMBDAcI:Term	5.0uL	0.5uL	39.5uL	3.0uL	EcoRI	Xba1
L6b, Pro:LAMBDAcI:Term	5.0uL	0.5uL	41.5uL	1.0uL	EcoRI	Xba1
L6e, Pro:LAMBDAcI:Term	5.0uL	0.5uL	39.5uL	3.0uL	EcoRI	Xba1
L6d, Pro:LAMBDAcI:Term	5.0uL	0.5uL	39.5uL	3.0uL	EcoRI	Xba1
L7a, Pro:LAMBDAcI:Term	5.0uL	0.5uL	38.5uL	4.0uL	EcoRI	Xba1
L7b, Pro:LAMBDAcI:Term	5.0uL	0.5uL	39.5uL	3.0uL	EcoRI	Xba1
L7d, Pro:LAMBDAcI:Term	5.0uL	0.5uL	37.5uL	5.0uL	EcoRI	Xba1
L8a, Pro:LAMBDAcI:Term	5.0uL	0.5uL	39.5uL	3.0uL	EcoRI	Xba1
L9a, Pro:LAMBDAcI:Term	5.0uL	0.5uL	41.5uL	1.0uL	EcoRI	Xba1
L9c, Pro:LAMBDAcI:Term	5.0uL	0.5uL	40.5uL	2.0uL	EcoRI	Xba1
L9d, Pro:LAMBDAcI:Term	5.0uL	0.5uL	41.5uL	1.0uL	EcoRI	Xba1

@@ Line 14: / Line 14: @@
 |3. '''Sequence L5, L6-L9'''
 |-
-|4. '''Re-transform RFP, TetR promoter, terminator''' because no cell growth on plates.
+|4. '''Re-transform RFP, TetR promoter, terminator, and MCherry''' because no cell growth on plates. Place in 2mL LB cultures, allow growth in incubator @37C for 2 hours. Plate samples on Ampicillin resistant plates. Allow growth O/N.
 |-
 |5. '''Discuss problems with sequences'''