Minnesota/22 July 2008

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Latest revision as of 23:12, 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: RE stands for restriction enzyme. 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

Parts	10x Buffer	H20	BaseVector	Insert 1	Insert 2	T4 DNA ligase
(a) Lac/LAMBDAcI:GFP:term	3.0uL	6.0uL	0	15.0uL Lac/LAMBDAcI	5.0uL GFP:term	1.0uL
(b) Lac/LAMBDAcI:GFP:term	3.0uL	6.0uL	0	15.0uL Lac/LAMBDAcI	5.0uL GFP:term	1.0uL
LacIpro:p22cII:BV	3.0uL	15.5uL	2.5uL	0.5uL LacIpro.	7.5uL p22cII	1.0uL
Tet/p22:R2:BV	3.0uL	8.5uL	2.5uL	7.5uL Tet/p22	7.5uL RFP(R2)	1.0uL
Tet/p22:Y+2:BV	3.0uL	8.5uL	2.5uL	7.5uL Tet/p22	7.5uL YFPw/LVAtag(Y+2)	1.0uL
Tet/p22:Y+4:BV	3.0uL	8.5uL	2.5uL	7.5uL Tet/p22	7.5uL YFPw/LVAtag(Y+4)	1.0uL
Tet/p22:R+4:BV	3.0uL	8.5uL	2.5uL	7.5uL Tet/p22	7.5uL RFPw/LVAtag(R+4)	1.0uL

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: PROBLEM WITH SEQUENCING RESULTS. The ccdb gene still exists, but didn't terminate the E. Coli. This means the ccdb gene is either mutated or doesn't work properly. Solution: Pick 2 new base vector (DB3.1 E.Coli with ccdb gene) colonies in hope that the 2 new colonies will not have the same mutated/malfunctioned ccdb gene, and starting 2 new 50.0mL cultures to incubate O/N, from these cultures we will maxi-prep thus purifying out/extracting the DNA from the E. Coli and thus using this extracted plasmid/DNA to then use as the backbone.

@@ Line 15: / Line 15: @@
 |-
 |'''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:
 {|border="1" align="left"
@@ Line 36: / Line 37: @@
 |-
-|'''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.
+|''NOTE:'' RE stands for restriction enzyme. 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:
+{|border="1" align="left"
+|-
+!| 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:
@@ Line 47: / Line 64: @@
 |-
 |'''d.''' LacI Promoter + p22 cII + BV
+{|border="1" align="left"
+|-
+!| Parts !! 10x Buffer !! H20 !! BaseVector !! Insert 1 !! Insert 2 !! T4 DNA ligase
+|-
+|(a) Lac/LAMBDAcI:GFP:term || 3.0uL || 6.0uL || 0  || 15.0uL Lac/LAMBDAcI || 5.0uL GFP:term || 1.0uL
+|-
+|(b) Lac/LAMBDAcI:GFP:term || 3.0uL || 6.0uL || 0 || 15.0uL Lac/LAMBDAcI || 5.0uL GFP:term || 1.0uL
+|-
+| LacIpro:p22cII:BV || 3.0uL || 15.5uL || 2.5uL || 0.5uL LacIpro. || 7.5uL p22cII || 1.0uL
+|-
+| Tet/p22:R2:BV || 3.0uL || 8.5uL || 2.5uL || 7.5uL Tet/p22 || 7.5uL RFP(R2) || 1.0uL
+|-
+| Tet/p22:Y+2:BV || 3.0uL || 8.5uL || 2.5uL || 7.5uL Tet/p22 || 7.5uL YFPw/LVAtag(Y+2) || 1.0uL
+|-
+| Tet/p22:Y+4:BV || 3.0uL || 8.5uL || 2.5uL || 7.5uL Tet/p22 || 7.5uL YFPw/LVAtag(Y+4) || 1.0uL
+|-
+| Tet/p22:R+4:BV || 3.0uL || 8.5uL || 2.5uL || 7.5uL Tet/p22 || 7.5uL RFPw/LVAtag(R+4) || 1.0uL
+|}
 |-
 |'''7. Transformation:''' Procedure performed in 30 minutes. Transform all ligation products. Incubate in 2mL LB cultures for 2 hours @37C with shaking @ 220rpm's.
@@ Line 52: / Line 91: @@
 |'''8. Plate transformations:''' Plate transformation cultures.
 |-
-|'''9. Prepare sequencing reactions:''' Prepare sequencing rxns IF POSSIBLE.
+|'''9. Prepare sequencing reactions:''' PROBLEM WITH SEQUENCING RESULTS. The ccdb gene still exists, but didn't terminate the E. Coli. This means the ccdb gene is either mutated or doesn't work properly. ''Solution:'' Pick 2 new base vector (DB3.1 E.Coli with ccdb gene) colonies in hope that the 2 new colonies will not have the same mutated/malfunctioned ccdb gene, and starting 2 new 50.0mL cultures to incubate O/N, from these cultures we will maxi-prep thus purifying out/extracting the DNA from the E. Coli and thus using this extracted plasmid/DNA to then use as the backbone.