Minnesota/22 July 2008

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Revision as of 21:09, 22 July 2008

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1. Pick Colonies from plates made 07-21-2008 Start cultures.

2. Plasmid prep: Prep RFP, YFP, GFP, TetR promoter, Terminator. Follow QIA miniprep procedure --> 1hr long.

3. Spec the prep products: Using spectrophotometry, the DNA concentration of the plasmid prep products were measured.

4. Double digest: Follow Kat's DNA work procedure to perform Double digest on: LacI Promoter, p22 cII gene, RFP, YFP, BV (dual promoters, GFP:Term already d.dig.). Incubate digested products for 2 hours @37C. Heat inactivate digestion enzyme for 15 mins @ 65C water bath.

5. Vector Dephosphorylation: Same dephos. procedure used on GFP:Terminator, BV. After dephosphorylation, incubate @37C for 30 mins. Heat inactivate dephosphorylation enzyme for 15 mins in 65C water bath.

6. Ligation Reactions: Procedure performed in 20 minutes. Once all were ligated, were then incubated @ 16C for 1 hour. Heat inactivated enzyme @ 65C for 15 minutes. Ligated the following using L4 DNA Ligase:

a. BV + TetR:p22 promoter + RFP

b. BV + TetR:p22 promoter + YFP

c. LacI:LambdacI + GFP:Terminator

d. LacI Promoter + p22 cII + BV

7. Transformation: Procedure performed in 30 minutes. Transform all ligation products. Incubate in 2mL LB cultures for 2 hours @37C with shaking @ 220rpm's.

8. Plate transformations: Plate transformation cultures.

9. Prepare sequencing reactions: Prepare sequencing rxns IF POSSIBLE.

@@ Line 11: / Line 11: @@
 |-
 |'''2. Plasmid prep:'''  Prep RFP, YFP, GFP, TetR promoter, Terminator. Follow QIA miniprep procedure --> 1hr long.
+|-
+|'''3. Spec the prep products:''' Using spectrophotometry, the DNA concentration of the plasmid prep products were measured.
 |-
-|'''3. Double digest:''' Follow Kat's DNA work procedure to perform Double digest on: LacI Promoter, p22 cII gene, RFP, YFP, BV (dual promoters, GFP:Term already d.dig.). Incubate digested products for 2 hours @37C. Heat inactivate digestion enzyme for 15 mins @ 65C water bath.
+|'''4. Double digest:''' Follow Kat's DNA work procedure to perform Double digest on: LacI Promoter, p22 cII gene, RFP, YFP, BV (dual promoters, GFP:Term already d.dig.). Incubate digested products for 2 hours @37C. Heat inactivate digestion enzyme for 15 mins @ 65C water bath.
 |-
-|'''4. Vector Dephosphorylation:''' Same dephos. procedure used on GFP:Terminator, BV. After dephosphorylation, incubate @37C for 30 mins. Heat inactivate dephosphorylation enzyme for 15 mins in 65C water bath.
+|'''5. Vector Dephosphorylation:''' Same dephos. procedure used on GFP:Terminator, BV. After dephosphorylation, incubate @37C for 30 mins. Heat inactivate dephosphorylation enzyme for 15 mins in 65C water bath.
 |-
-|'''5. Ligation Reactions:''' Procedure performed in 20 minutes. Once all were ligated, were then incubated @ 16C for 1 hour. Heat inactivated enzyme @ 65C for 15 minutes. Ligated the following using L4 DNA Ligase:
+|'''6. Ligation Reactions:''' Procedure performed in 20 minutes. Once all were ligated, were then incubated @ 16C for 1 hour. Heat inactivated enzyme @ 65C for 15 minutes. Ligated the following using L4 DNA Ligase:
 |-
 |'''a.''' BV + TetR:p22 promoter + RFP
@@ Line 26: / Line 28: @@
 |'''d.''' LacI Promoter + p22 cII + BV
 |-
-|'''6. Transformation:''' Procedure performed in 30 minutes. Transform all ligation products. Incubate in 2mL LB cultures for 2 hours @37C with shaking @ 220rpm's.
+|'''7. Transformation:''' Procedure performed in 30 minutes. Transform all ligation products. Incubate in 2mL LB cultures for 2 hours @37C with shaking @ 220rpm's.
 |-
-|'''7. Plate transformations:''' Plate transformation cultures.
+|'''8. Plate transformations:''' Plate transformation cultures.
 |-
-|'''8. Prepare sequencing reactions:''' Prepare sequencing rxns IF POSSIBLE.
+|'''9. Prepare sequencing reactions:''' Prepare sequencing rxns IF POSSIBLE.