Minnesota/9 July 2008

From 2008.igem.org

(Difference between revisions)

Latest revision as of 19:21, 10 July 2008

Back to Notebook Home
Go to Previous Day (July 8)	Go to Next Day (July 10)

1. Single Digests: Performed today. Incubated digested samples for 2hrs @ 37C. Refer to table below.

Gene	10x Buffer	BSA Protein	H20	DNA	RE
Promoter	5.0uL	0.5uL	40.4uL	2.1uL	2.0uL, SPE
LAMBDAcI	5.0uL	0.5uL	29.5uL	15.0uL	1.0uL, XBA 1
LAMBDAcI	5.0uL	0.5uL	29.5uL	15.0uL	1.0uL, SPE
Terminator	5.0uL	0.5uL	38.0uL	4.5uL	2.0uL, XBA 1

NOTE: RE = Restriction Enzyme

2. Ligation: Performed today. Heat inactivated enzyme @ 65C for 15 min to kill enzymes.Ligate samples. Incubate ligated samples @ 16C for 1hour. Refer to table

below.

Genes	10x Buffer	H20	Base Vector	Insert	DNA Ligase
LAMBDAcI/Terminator	2.0uL	10.7uL	2.0uL (Term.)	4.3uL (LAMBDAcI)	1.0uL
Promoter/LAMBDAcI	2.0uL	10.7uL	2.0uL (Pro.)	4.3uL (LAMBDAcI)	1.0uL

3. Double Digests: Performed today. After digestion, incubate samples for 2hrs @37C. Heat inactivate restriction site enzymes EcorI and PstI in a water bath @ 65C for 15 minutes to denature the enzymes, thus stopping/controlling digestion. Place digested samples in -20C freezer overnight.

Gene	10x Buffer	BSA Protein	H20	DNA	RE 1	RE 2
Promoter/LAMBDAcI	5.0uL	0.5uL	22.5uL	20.0uL	1.0uL, EcorI	1.0uL, PstI
LAMBDAcI/Terminator	5.0uL	0.5uL	20.0uL	20.0uL	1.0uL, EcorI	1.0uL, PstI
Base Vector	5.0uL	0.5uL	15.0uL	15.0uL	1.0uL, EcorI	1.0uL, PstI

NOTE: RE = Restriction Enzyme

4. Make/Pour Ampicillin Plates

5. Order Primers for Part Sequencing

6. Purified Plasmid Prep: Purified MCherry, Promoter, Terminator and Base Vector because sequencing results were poor.

@@ Line 5: / Line 5: @@
 |'''[[Minnesota/8 July 2008|Go to Previous Day (July 8)]]'''|| width=158|'''[[Minnesota/10 July 2008|Go to Next Day (July 10)]]'''
 |}
 {|
@@ Line 26: / Line 27: @@
 |-
 |NOTE: RE = Restriction Enzyme
 |-
-|2. '''Ligation''': Performed today. Incubate ligated samples @ 16C for 1hour. Heat inactivated enzyme @ 65C for 15 min. Refer to table below.
+|2. '''Ligation''': Performed today. Heat inactivated enzyme @ 65C for 15 min to kill enzymes.Ligate samples. Incubate ligated samples @ 16C for 1hour. Refer to table
+below.
 {|border="1" align="left"
@@ Line 43: / Line 44: @@
 |-
-|3. '''Double Digests'''
+|3. '''Double Digests''': Performed today. After digestion, incubate samples for 2hrs @37C. Heat inactivate restriction site enzymes EcorI and PstI in a water bath @ 65C for 15 minutes to denature the enzymes, thus stopping/controlling digestion. Place digested samples in -20C freezer overnight.
+{|border="1" align="left"
+|-
+!| Gene !!10x Buffer !!BSA Protein !!H20 !!DNA  !!RE 1 !!RE 2
+|-
+|Promoter/LAMBDAcI ||5.0uL ||0.5uL ||22.5uL ||20.0uL ||1.0uL, EcorI ||1.0uL, PstI
+|-
+|LAMBDAcI/Terminator  ||5.0uL ||0.5uL ||20.0uL ||20.0uL ||1.0uL, EcorI ||1.0uL, PstI
+|-
+|Base Vector ||5.0uL ||0.5uL ||15.0uL ||15.0uL ||1.0uL, EcorI ||1.0uL, PstI
+|-
+|}
+|-
+|NOTE: RE = Restriction Enzyme
 |-
 |4. '''Make/Pour Ampicillin Plates'''
 |-
+|5. '''Order Primers for Part Sequencing'''
+|-
+|6. '''Purified Plasmid Prep''': Purified MCherry, Promoter, Terminator and Base Vector because sequencing results were poor.
 |}