Minnesota/22 July 2008

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Revision as of 21:45, 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. Follow the table below for double digest guidelines:

Parts	10x Buffer	BSA	H20	DNA	RE 1	RE 2
R2	5.0uL	0.5uL	40.5uL	2.0uL	1.0uL, Xba1	1.0uL, Pst1
Y+2	5.0uL	0.5uL	40.5uL	2.0uL	1.0uL, Xba1	1.0uL, Pst1
Y+4	5.0uL	0.5uL	39.5uL	3.0uL	1.0uL, Xba1	1.0uL, Pst1
R+4	5.0uL	0.5uL	9.5uL	33.0uL	1.0uL, Xba1	1.0uL, Pst1
Lac Pro.	5.0uL	0.5uL	41.5uL	1.0uL	1.0uL, EcoRI	1.0uL, Spe1
p22 cII	5.0uL	0.5uL	39.5uL	3.0uL	1.0uL, Xba1	1.0uL, Pst1
BV	5.0uL	0.5uL	9.5uL	33.0uL	1.0uL, EcoRI	1.0uL, Pst1

NOTE: Parts chosen that had good 'spec' results, meaning there is a high concentration of DNA. R2 = RFP, Y+2 = YFP with LVA tag, Y+4 = YFP with LVA tag, R+4 = RFP with LVA tag, Lac Pro. = LacI repressed promoter, p22 cII = gene for p22 cII, BV = base vector. DNA added into each one correlates with which part is being used, for example: R2 would have 2.0uL of RFP, and Y+2 would have 2.0uL of YFP with LVA tag, etc..

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

Parts	Antarctic phosphotase (enzyme)	Antarctic phosphotase Buffer
GFP:Terminator	Dephos. enzyme, 1.0uL	5.6uL
BV	Dephos. enzyme, 1.0uL	5.6uL

NOTE: Dephosphorylating enzyme and buffer were added into BV tube and GFP:Terminator tube, so no DNA had to be measured since was being put into that particular DNA tube already.

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.

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-|NOTE: Dephosphorylating enzyme and buffer were added into BV tube and GFP:Terminator tube, so no DNA had to be measured since was being put into that particular DNA tube already.
+|''NOTE:'' Dephosphorylating enzyme and buffer were added into BV tube and GFP:Terminator tube, so no DNA had to be measured since was being put into that particular DNA tube already.
 |-
 |'''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: