Minnesota/25 July 2008

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Latest revision as of 21:06, 6 August 2008

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1. No growth on plates: Experiments on 07-24-08 failed to show growth on Kanamycin resistant plates. Russian roulette anyone?

2. Proposed Schedule for today: 10:40-12:40: BV Digest...12:40-12:55: Restriction Enzyme inactivation...1:15-1:45: BV Dephosphorylation...1:45-1:55: Phosphatase enzyme inactivation...2:15-2:45: Ligation @ 16C...2:45-3:00: Ligase enzyme inactivation...3:00-3:45: Transformation...5:45: Plating on kan. res. plates.

3. Double Digest: Redo double digest of Base Vector. Follow the table below:

Parts	10x Buffer	BSA	H20	Insert DNA	RE 1	RE 2
Base Vector	5.0uL	0.5uL	39.5uL	3.0uL	1.0uL, EcoRI	1.0uL, Pst1

4. Redoing ligation: Using left over double digest from 07-24-08, But increase the amount of ligase in the reaction and performing the ligations @ 60C which is the optimal temperature for the ligase.

Parts	10x Buffer	H20	Base Vector	Insert DNA	T4 DNA Ligase	Total Volume
BV + Tet1	4.0uL	14.0uL	1.0uL	17.0uL	4.0uL	40.0uL
BV + Lac2	4.0uL	16.0uL	1.0uL	15.0uL	4.0uL	40.0uL
BV + Lac/LAMBDA	4.0uL	18.0uL	1.0uL	13.0uL	4.0uL	40.0uL
BV + Tet/p22	4.0uL	14.0uL	1.0uL	17.0uL	4.0uL	40.0uL

5. Redoing transformations: Redo with new ligated products by adding much less DNA/plasmid to the TOP10 Cells, b/c if put too much ligated DNA then the cells do not transform properly (as stated by iGEM troubleshooting). Add transformed products to 250uL of SOC broth, since this type of broth is ideal for TOP10 Cells. Warm SOC broth in tubes in an incubator before adding transformed products (product + TOP10 Cells).

@@ Line 3: / Line 3: @@
 |'''[[Team:Minnesota/NotebookComparator|  Back to Notebook Home]]'''
 |-
-|'''[[Minnesota/24 July 2008|Go to Previous Day (July 24)]]'''|| width=158|'''[[Minnesota/28 July 2008|Go to Next Day (July 28)]]'''
+|'''[[Minnesota/24 July 2008|Go to Previous Day (July 24)]]'''|| width=158|'''[[Minnesota/26 July 2008|Go to Next Day (July 26)]]'''
 |}
 {|
 |-
-|'''1. No growth on plates:''' Experiments on 07-24-08 failed to show growth on Kanamycin resistant plates.
+|'''1. No growth on plates:''' Experiments on 07-24-08 failed to show growth on Kanamycin resistant plates. Russian roulette anyone?
 |-
-|'''2. Redoing ligation:''' Using left over double digest from 07-24-08, But increase the amount of ligase in the reaction and performing the ligations @ 60C which is the optimal temperature for the ligase.
+|'''2. Proposed Schedule for today:''' 10:40-12:40: BV Digest...12:40-12:55: Restriction Enzyme inactivation...1:15-1:45: BV Dephosphorylation...1:45-1:55: Phosphatase enzyme inactivation...2:15-2:45: Ligation @ 16C...2:45-3:00: Ligase enzyme inactivation...3:00-3:45: Transformation...5:45: Plating on kan. res. plates.
 |-
-|'''3. Redoing transformations:''' Redo with new ligated products by adding much less DNA/plasmid to the TOP10 Cells, b/c if put too much ligated DNA then the cells do not transform properly (as stated by iGEM troubleshooting).
+|'''3. Double Digest:''' Redo double digest of Base Vector. Follow the table below:
+{|border="1" align="left"
+|-
+!| Parts !! 10x Buffer !! BSA !! H20 !! Insert DNA !! RE 1 !! RE 2
+|-
+| Base Vector || 5.0uL || 0.5uL || 39.5uL || 3.0uL || 1.0uL, EcoRI || 1.0uL, Pst1
+|}
+|-
+|'''4. Redoing ligation:''' Using left over double digest from 07-24-08, But increase the amount of ligase in the reaction and performing the ligations @ 60C which is the optimal temperature for the ligase.
+{|border="1" align="left"
+|-
+!| Parts !! 10x Buffer !! H20 !! Base Vector !! Insert DNA !! T4 DNA Ligase !! Total Volume
+|-
+| BV + Tet1 || 4.0uL || 14.0uL || 1.0uL || 17.0uL || 4.0uL || 40.0uL
+|-
+| BV + Lac2 || 4.0uL || 16.0uL || 1.0uL || 15.0uL || 4.0uL || 40.0uL
+|-
+| BV + Lac/LAMBDA || 4.0uL || 18.0uL || 1.0uL || 13.0uL || 4.0uL || 40.0uL
+|-
+| BV + Tet/p22 || 4.0uL || 14.0uL ||1.0uL || 17.0uL || 4.0uL || 40.0uL
+|}
+|-
+|'''5. Redoing transformations:''' Redo with new ligated products by adding much less DNA/plasmid to the TOP10 Cells, b/c if put too much ligated DNA then the cells do not transform properly (as stated by iGEM troubleshooting). Add transformed products to 250uL of SOC broth, since this type of broth is ideal for TOP10 Cells. Warm SOC broth in tubes in an incubator before adding transformed products (product + TOP10 Cells).