Minnesota/22 July 2008

From 2008.igem.org

(Difference between revisions)

Revision as of 20:58, 21 July 2008

Back to Notebook Home
Go to Previous Day (July 21)	Go to Next Day (July 23)

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. 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. 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

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. Plate transformations: Plate transformation cultures.

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

@@ Line 26: / Line 26: @@
 |'''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. Plate transformations:''' Plate transformation cultures.
+|-
+|'''8. Prepare sequencing reactions:''' Prepare sequencing rxns IF POSSIBLE.