Minnesota/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

7. Gel Electrophoresis: Performed on double digest reaction from above. Refer to picture below for results.

Gel from 07-17-2008

Left to Right: Ladder, --, L9E, L9C, L9A, L8A, L7D, L7B, L7A, L6E, L6D, L6B, L6A, L5C, --, Ladder, L4j, L4i, L4h, L4g, L4d, L4e, L4c, L4b, L3j, L3i, L2b, L2a, --, Ladder

@@ Line 18: / Line 18: @@
 |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:
+|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:
 {|border="1" align="left"
@@ Line 48: / Line 49: @@
 |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
+|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
@@ Line 79: / Line 80: @@
 |[[Image:7.17.08gel.jpg|thumb|center|600px|Gel from 07-17-2008]]
 |-
-|Left to Right: Ladder, --, L9E, L9C, L9A, L8A, L7D, L7B, L7A, L6E, L6D, L6B, L6A, L5C, --, Ladder, L4j, L4i, L4h, L4g, L4d, L4e, L4c, L4b, L3j, L3i, L2b, L2a, --, Ladder
+|''Left to Right:'' Ladder, --, L9E, L9C, L9A, L8A, L7D, L7B, L7A, L6E, L6D, L6B, L6A, L5C, --, Ladder, L4j, L4i, L4h, L4g, L4d, L4e, L4c, L4b, L3j, L3i, L2b, L2a, --, Ladder
 |-
 |}