Refactored Decaffeination Operon

From 2012.igem.org

(Difference between revisions)
(Created page with "Experiment 2: Constructon of a refactored decaffeination operon 2a. Phusion PCR Primers: EQ_pSB1c3_NdmA_for: TAGGTACAGTGCTAGCTACTAGAGAAATCAAATTAAGGAGGTAAGATAAATGGAACAGGCAATCA...")
(→Experiment 2: Construction of a refactored decaffeination operon)
 
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Experiment 2: Constructon of a refactored decaffeination operon
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2a. Phusion PCR
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Primers:
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==Experiment 2: Construction of a refactored decaffeination operon==
-
EQ_pSB1c3_NdmA_for: TAGGTACAGTGCTAGCTACTAGAGAAATCAAATTAAGGAGGTAAGATAAATGGAACAGGCAATCATTAATGATGAACGGG
+
-
EQ_pBSC1C_NdmA_2: TGATTTCTGGAATTCGCGGCCGCTTCTAGAGATTAAGGAGGTAAGATAAATGGAACAGGCAATCATTAATGATGAACGGG
+
-
EQ_pSB1C3_Gib_rev: TTGATTTCTCTAGTAGCTAGCACTGTACCTAGGACTG
+
-
EQ_NdmA_rev: TTATATGTAGCTCCTATCGCTTTCAATGACTGGG
+
-
EQ_RBS_NdmB_for: gtCATTGAAAGCGATAGGAGCTACATATAATCTAGAGAAAGAGGAGAAATACTAGATGAAAGAACAGCTCAAGCCGCTG
+
-
EQ_NdmB_rev: ccggtctcgcTTACTGTTCTTCTTCAATAACATTCGTCAAGACG
+
-
EQ_RBS_NdmC_for: GAAGAAGAACAGTAAgcgagaccggTCTAGAGAAAGAGGAGAAATACTAGATGTCTACTGACCAAGTAATTTTTAACGactgg
+
-
EQ_NdmC_rev: TTAGTCCCGCAGAGCACCATATTGCac
+
-
EQ_RBS_NdmD for: GCAATATGGTGCTCTGCGGGACTAATCTAGAGAAAGAGGAGAAATACTAGATGAACAAACTTGACGTCAACCagtgg
+
-
EQ_NdmD_pBS1C_rev: TTATACAGCTCGTCCATACCGTGGGTGATGCCCGGCCTCACAGATCGAGAACGATT
+
-
EQ_pBS1C_Gib_for: GGGCATCACCCACGGTATGGACGAG
+
-
EQ_pBS1C_rev_2: CTCTAGAAGCGGCCGCGAATTCCAGAAATCA
+
 +
2a.  Phusion PCR<br/>
 +
Primers:<br/>
 +
EQ_pSB1c3_NdmA_for: TAGGTACAGTGCTAGCTACTAGAGAAATCAAATTAAGGAGGTAAGATAAATGGAACAGGCAATCATTAATGATGAACGGG<br/>
 +
EQ_pBSC1C_NdmA_2: TGATTTCTGGAATTCGCGGCCGCTTCTAGAGATTAAGGAGGTAAGATAAATGGAACAGGCAATCATTAATGATGAACGGG<br/>
 +
EQ_pSB1C3_Gib_rev: TTGATTTCTCTAGTAGCTAGCACTGTACCTAGGACTG<br/>
 +
EQ_NdmA_rev: TTATATGTAGCTCCTATCGCTTTCAATGACTGGG<br/>
 +
EQ_RBS_NdmB_for: gtCATTGAAAGCGATAGGAGCTACATATAATCTAGAGAAAGAGGAGAAATACTAGATGAAAGAACAGCTCAAGCCGCTG<br/>
 +
EQ_NdmB_rev: ccggtctcgcTTACTGTTCTTCTTCAATAACATTCGTCAAGACG<br/>
 +
EQ_RBS_NdmC_for: GAAGAAGAACAGTAAgcgagaccggTCTAGAGAAAGAGGAGAAATACTAGATGTCTACTGACCAAGTAATTTTTAACGactgg<br/>
 +
EQ_NdmC_rev: TTAGTCCCGCAGAGCACCATATTGCac<br/>
 +
EQ_RBS_NdmD for: GCAATATGGTGCTCTGCGGGACTAATCTAGAGAAAGAGGAGAAATACTAGATGAACAAACTTGACGTCAACCagtgg<br/>
 +
EQ_NdmD_pBS1C_rev: TTATACAGCTCGTCCATACCGTGGGTGATGCCCGGCCTCACAGATCGAGAACGATT<br/>
 +
EQ_pBS1C_Gib_for: GGGCATCACCCACGGTATGGACGAG<br/>
 +
EQ_pBS1C_rev_2: CTCTAGAAGCGGCCGCGAATTCCAGAAATCA<br/>
-
10 uL 5x Phusion HF Buffer
 
-
1 uL 10mM dNTPs
 
-
5 uL 5uM forward primer
 
-
5 uL 5uM reverse primer
 
-
1 uL DMSO
 
-
1 uL template
 
-
26.5 uL H20
 
-
--
 
-
.5 uL phusion polymerase
 
-
rxns:
 
-
Vector PCRs
+
10 uL 5x Phusion HF Buffer<br/>
-
1. Bba_k515105  + EQ_pSB1C3_Gib_for + EQ_pSB1c3_gib_rev
+
1 uL 10mM dNTPs<br/>
-
2. Bba_k515105  + EQ_pSB1C3_Gib_for + EQ_pSB1c3_gib_rev_2
+
5 uL 5uM forward primer<br/>
 +
5 uL 5uM reverse primer<br/>
 +
1 uL DMSO<br/>
 +
1 uL template<br/>
 +
26.5 uL H20<br/>
 +
--<br/>
 +
.5 uL phusion polymerase<br/>
-
Insert PCRs
+
rxns:<br/>
-
3. CBB5 cells + EQ_NdmA_for + EQ_NdmA_rev
+
-
4. CBB5 cells + EQ_pSB1C3_NdmA_2 + EQ_NdmA_rev
+
-
5. CBB5 cells + EQ_RBS_NdmB_for + EQ_NdmB_rev
+
-
6. CBB5 cells + EQ_RBS_NdmC_for + EQ_NdmC_rev
+
-
7. CBB5 cells + EQ_RBS_NdmD_for + EQ_NdmD_pSB1C_rev
+
-
PCR cycling:
+
Vector PCRs<br/>
 +
1. Bba_k515105  + EQ_pSB1C3_Gib_for + EQ_pSB1c3_gib_rev<br/>
 +
2. Bba_k515105  + EQ_pSB1C3_Gib_for + EQ_pSB1c3_gib_rev_2<br/>
-
98 deg x 3 min
+
Insert PCRs<br/>
-
--
+
3. CBB5 cells + EQ_NdmA_for + EQ_NdmA_rev<br/>
-
98 deg x 30s
+
4. CBB5 cells + EQ_pSB1C3_NdmA_2 + EQ_NdmA_rev<br/>
-
58 deg x 20s      x30 cycles
+
5. CBB5 cells + EQ_RBS_NdmB_for + EQ_NdmB_rev<br/>
-
72 deg x 2 min
+
6. CBB5 cells + EQ_RBS_NdmC_for + EQ_NdmC_rev<br/>
-
--
+
7. CBB5 cells + EQ_RBS_NdmD_for + EQ_NdmD_pSB1C_rev<br/>
-
72 deg x 10 min
+
 +
PCR cycling:<br/>
-
Gel igm2a
+
98 deg x 3 min<br/>
 +
--<br/>
 +
98 deg x 30s<br/>
 +
58 deg x 20s      x30 cycles<br/>
 +
72 deg x 2 min<br/>
 +
--<br/>
 +
72 deg x 10 min<br/>
 +
Run 1 uL 2a1-7, 3.5 uL Fermentas 1kb GeneRuler ladder
-
2b. PCR purification
+
[[File:2a.jpg]]
-
Qiagen PCR purification protocol
 
-
Elute in 35 uL h20
 
-
nanodrop concentrations:
+
2b. PCR purification<br/>
-
1. vector 1: 289.0 ng/uL
+
Qiagen PCR purification protocol<br/>
-
2. vector 2: 85.6 ng/uL
+
-
3. NdmA_1: 139.8 ng/uL
+
-
4. NdmA_2: 144.5 ngl/uL
+
-
5. NdmB: 144.6ng/uL
+
-
6. NdmC: 146.1 ng/uL
+
-
7. NdmD: 104.8ng/uL
+
-
2c: DpnI digest of vector PCRs
+
Elute in 35 uL h20<br/>
-
1.
+
nanodrop concentrations:<br/>
-
15 uL vector 1
+
-
5 uL 10x NEB 4 Buffer
+
-
1 uL dpnI
+
-
29 uL H20
+
-
--
+
-
50 uL
+
-
2.
+
1. vector 1: 289.0 ng/uL<br/>
-
30 uL vector 2
+
2. vector 2: 85.6 ng/uL<br/>
-
5 uL 10x NEB 4 Buffer
+
3. NdmA_1: 139.8 ng/uL<br/>
-
1 uL dpnI
+
4. NdmA_2: 144.5 ngl/uL<br/>
-
14 uL H20
+
5. NdmB: 144.6ng/uL<br/>
-
--
+
6. NdmC: 146.1 ng/uL<br/>
-
50 uL
+
7. NdmD: 104.8ng/uL<br/>
-
37 deg x 2 hrs
+
2c: DpnI digest of vector PCRs<br/>
-
2e. Purification
+
1.<br/>
 +
15 uL vector 1<br/>
 +
5 uL 10x NEB 4 Buffer<br/>
 +
1 uL dpnI<br/>
 +
29 uL H20<br/>
 +
--<br/>
 +
50 uL <br/>
-
Standard Qiagen PCR purification protocol. Elute in H20.
+
2.<br/>
 +
30 uL vector 2<br/>
 +
5 uL 10x NEB 4 Buffer<br/>
 +
1 uL dpnI<br/>
 +
14 uL H20<br/>
 +
--<br/>
 +
50 uL<br/>
 +
37 deg x 2 hrs<br/>
 +
2e. Purification<br/>
-
2F. Gibson cloning
+
Standard Qiagen PCR purification protocol. Elute in H20.<br/>
-
pmols = weight(ng) x 1000 / (bp x 650 daltons)
 
-
Will use .05 pmol of vector, .1 pmol for each insert
+
2F. Gibson cloning<br/>
-
Vec 1 (.05pmol) = need 68.57ng / (79.5ng/uL) = .86uL
+
pmols = weight(ng) x 1000 / (bp x 650 daltons)<br/>
-
Vec 2 (.05pmol) = need 68.57ng / (132.5ng/uL) = .52uL
+
-
NdmA1 (.1pmol) = need 71.5ng / (139.8ng/uL) = .51 uL
 
-
NdmA2 (.1pmol) = need 71.5ng / (144.5ng/uL) = .49 uL
 
-
NdmB  (.1pmol) = need 73.4ng / (144.6ng/uL) = .50 uL
 
-
NdmC  (.1pmol) = need 58.8ng / (146.1ng/uL) = .40 uL
 
-
NdmD  (.1pmol) = need 120.ng / (104.8ng/uL) = 1.15 uL
 
-
rxns:
+
Will use .05 pmol of vector, .1 pmol for each insert<br/>
-
1.
+
Vec 1 (.05pmol) = need 68.57ng / (79.5ng/uL) = .86uL<br/>
-
.86 uL vec 1
+
Vec 2 (.05pmol) = need 68.57ng / (132.5ng/uL) = .52uL<br/>
-
.51 uL ndmA_1
+
-
.50 uL NdmB
+
-
.40 uL NdmC
+
-
1.15 uL NdmD
+
-
15 ul 1.33x Gibson Master Mix
+
-
1.58 uL H20
+
-
--
+
-
20 uL total
+
-
2.
+
NdmA1 (.1pmol) = need 71.5ng / (139.8ng/uL) = .51 uL<br/>
-
.86 uL vec 1
+
NdmA2 (.1pmol) = need 71.5ng / (144.5ng/uL) = .49 uL<br/>
-
15 uL 1.33x Gibson Master Mix
+
NdmB  (.1pmol) = need 73.4ng / (144.6ng/uL) = .50 uL<br/>
-
4.14 uL H20
+
NdmC  (.1pmol) = need 58.8ng / (146.1ng/uL) = .40 uL<br/>
-
--
+
NdmD  (.1pmol) = need 120.ng / (104.8ng/uL) = 1.15 uL<br/>
-
20 uL total
+
-
3.
+
rxns:<br/>
-
.52 uL Vec 2
+
-
.51 uL ndmA_1
+
-
.50 uL NdmB
+
-
.40 uL NdmC
+
-
1.15 uL NdmD
+
-
15 ul 1.33x Gibson Master Mix
+
-
1.94 uL H20
+
-
--
+
-
20 uL total
+
-
4.
+
1.<br/>
-
.52 uL vec 1
+
.86 uL vec 1<br/>
-
15 uL 1.33x Gibson Master Mix
+
.51 uL ndmA_1<br/>
-
4.14 uL H20
+
.50 uL NdmB<br/>
-
--
+
.40 uL NdmC<br/>
-
20 uL total
+
1.15 uL NdmD<br/>
 +
15 ul 1.33x Gibson Master Mix<br/>
 +
1.58 uL H20<br/>
 +
--<br/>
 +
20 uL total<br/>
-
50 deg thermocycler x 60 minutes
+
2.<br/>
 +
.86 uL vec 1<br/>
 +
15 uL 1.33x Gibson Master Mix<br/>
 +
4.14 uL H20<br/>
 +
--<br/>
 +
20 uL total<br/>
-
Run 5 uL each reaction on .8% agarose gel
+
3.<br/>
 +
.52 uL Vec 2<br/>
 +
.51 uL ndmA_1<br/>
 +
.50 uL NdmB<br/>
 +
.40 uL NdmC<br/>
 +
1.15 uL NdmD<br/>
 +
15 ul 1.33x Gibson Master Mix<br/>
 +
1.94 uL H20<br/>
 +
--<br/>
 +
20 uL total<br/>
-
insert igm2f.jpeg
+
4.<br/>
 +
.52 uL vec 1<br/>
 +
15 uL 1.33x Gibson Master Mix<br/>
 +
4.14 uL H20<br/>
 +
--<br/>
 +
20 uL total<br/>
-
Desalted gibson reactions on .025uM Nitrocellulose membranes x 20 minutes
+
50 deg thermocycler x 60 minutes<br/>
-
2g. Electroporation
+
Run 5 uL each reaction (1-4)on .8% agarose gel + 3.5 ul Fermentas 1kb GeneRuler<br/>
-
Decided to not proceed with promoterless construct (2F3,4)
+
[[File:2f.jpg]]
-
1 uL desalted gibson reaction (2F1,2) ->50 uL electrocompetent BW25113-GuaB -> 1mL SOC -> 1hr incubation @ 37 deg
 
 +
Desalted gibson reactions on .025uM Nitrocellulose membranes x 20 minutes<br/>
-
2j.  Selective growth in Caffeine/Theophylline
+
2g.  Electroporation<br/>
-
dilute 60 uL 2g transformations (1-2) -> 3mL Mineral M9 media + 34ug/mL chloramphenicol +/- 100mg Caffeine or Theophylline.  Place on 30 deg shaker x 48 hrs
+
Decided to not proceed with promoterless construct (2F3,4)<br/>
 +
 
 +
1 uL desalted gibson reaction (2F1,2) ->50 uL electrocompetent BW25113-GuaB -> 1mL SOC -> 1hr incubation @ 37 deg<br/>
 +
 
 +
 
 +
2j.  Selective growth in Caffeine/Theophylline<br/>
 +
 
 +
dilute 60 uL 2g transformations (1-2) -> 3mL Mineral M9 media + 34ug/mL chloramphenicol +/- 100mg Caffeine or Theophylline.  Place on 30 deg shaker x 48 hrs<br/>
Results (growth):
Results (growth):
 +
{| cellpadding="5" border="1" cellspacing="0"
 +
! Media
 +
! 2g1 (+operon)
 +
! 2g2 (vector only)
 +
|-
 +
| M9
 +
|  -
 +
|  -
 +
|-
 +
| M9 + Caffeine
 +
|  -
 +
|  -
 +
|-
 +
| M9 + Theophylline
 +
|  +
 +
|  -
 +
|-
 +
|}
 +
 +
-
2g1 (+operon) 2g2 (vector only)
+
Result: construct enables growth (demethylation) on Theophylline. NdmC is not fuctional.<br/>
-
1. M9 - -
+
-
2. M9 + caffeine - -
+
-
3. M9 + Theophylline + _
+
-
Result: construct enables growth (demethylation) on Theophylline.  NdmC is not fuctional.
+
Streak 2j1 theophylline enriched culture onto LB-chloramphenicol plate for isolation of single colonies.<br/>
-
Streak 2j1 theophylline enriched culture onto LB-chloramphenicol plate for isolation of single colonies.
+
2K Plasmid Prep<br/>
-
2K Plasmid Prep
+
Pick 2 colonies from theophylline enriched streak for plasmid prep.  Prep 5mL culture.<br/>
-
Pick 2 colonies from theophylline enriched streak for plasmid prep.  Prep 5mL culture.
+
2L Restriction Digest<br/>
-
2L Restriction Digest
+
Perform restriction digest to analyze insert size of plasmids<br/>
-
Perform restriction digest to analyze insert size of plasmids
+
1 uL 2k1,2<br/>
 +
1 uL 10x NEB 3<br/>
 +
.5 uL NotI<br/>
 +
6.5 uL H20<br/>
 +
--<br/>
 +
10 uL<br/>
-
1 uL 2k1,2
+
37 deg x 1hr<br/>
-
1 uL 10x NEB 3
+
-
.5 uL NotI
+
-
6.5 uL H20
+
-
--
+
-
10 uL
+
-
37 deg x 1hr
+
Run 5 uL on .8% agarose gel<br/>
-
Run 5 uL on .8% agarose gel
+
[[File:2L.jpg|200px]]<br/>
-
insert gel image (on darkroom computer?)
+
Lanes: <br/>
 +
1. 2k1<br/>
 +
2. 2k2<br/>
 +
3. GeneRuler 1kb ladder
-
Result:  both clones show anticipated ~5kB band corresponding to the complete operon.
+
Result:  both clones show anticipated ~5kB band corresponding to the complete operon.<br/>

Latest revision as of 19:42, 20 August 2012


Experiment 2: Construction of a refactored decaffeination operon

2a. Phusion PCR

Primers:
EQ_pSB1c3_NdmA_for: TAGGTACAGTGCTAGCTACTAGAGAAATCAAATTAAGGAGGTAAGATAAATGGAACAGGCAATCATTAATGATGAACGGG
EQ_pBSC1C_NdmA_2: TGATTTCTGGAATTCGCGGCCGCTTCTAGAGATTAAGGAGGTAAGATAAATGGAACAGGCAATCATTAATGATGAACGGG
EQ_pSB1C3_Gib_rev: TTGATTTCTCTAGTAGCTAGCACTGTACCTAGGACTG
EQ_NdmA_rev: TTATATGTAGCTCCTATCGCTTTCAATGACTGGG
EQ_RBS_NdmB_for: gtCATTGAAAGCGATAGGAGCTACATATAATCTAGAGAAAGAGGAGAAATACTAGATGAAAGAACAGCTCAAGCCGCTG
EQ_NdmB_rev: ccggtctcgcTTACTGTTCTTCTTCAATAACATTCGTCAAGACG
EQ_RBS_NdmC_for: GAAGAAGAACAGTAAgcgagaccggTCTAGAGAAAGAGGAGAAATACTAGATGTCTACTGACCAAGTAATTTTTAACGactgg
EQ_NdmC_rev: TTAGTCCCGCAGAGCACCATATTGCac
EQ_RBS_NdmD for: GCAATATGGTGCTCTGCGGGACTAATCTAGAGAAAGAGGAGAAATACTAGATGAACAAACTTGACGTCAACCagtgg
EQ_NdmD_pBS1C_rev: TTATACAGCTCGTCCATACCGTGGGTGATGCCCGGCCTCACAGATCGAGAACGATT
EQ_pBS1C_Gib_for: GGGCATCACCCACGGTATGGACGAG
EQ_pBS1C_rev_2: CTCTAGAAGCGGCCGCGAATTCCAGAAATCA



10 uL 5x Phusion HF Buffer
1 uL 10mM dNTPs
5 uL 5uM forward primer
5 uL 5uM reverse primer
1 uL DMSO
1 uL template
26.5 uL H20
--
.5 uL phusion polymerase

rxns:

Vector PCRs
1. Bba_k515105 + EQ_pSB1C3_Gib_for + EQ_pSB1c3_gib_rev
2. Bba_k515105 + EQ_pSB1C3_Gib_for + EQ_pSB1c3_gib_rev_2

Insert PCRs
3. CBB5 cells + EQ_NdmA_for + EQ_NdmA_rev
4. CBB5 cells + EQ_pSB1C3_NdmA_2 + EQ_NdmA_rev
5. CBB5 cells + EQ_RBS_NdmB_for + EQ_NdmB_rev
6. CBB5 cells + EQ_RBS_NdmC_for + EQ_NdmC_rev
7. CBB5 cells + EQ_RBS_NdmD_for + EQ_NdmD_pSB1C_rev

PCR cycling:

98 deg x 3 min
--
98 deg x 30s
58 deg x 20s x30 cycles
72 deg x 2 min
--
72 deg x 10 min


Run 1 uL 2a1-7, 3.5 uL Fermentas 1kb GeneRuler ladder

2a.jpg


2b. PCR purification

Qiagen PCR purification protocol

Elute in 35 uL h20

nanodrop concentrations:

1. vector 1: 289.0 ng/uL
2. vector 2: 85.6 ng/uL
3. NdmA_1: 139.8 ng/uL
4. NdmA_2: 144.5 ngl/uL
5. NdmB: 144.6ng/uL
6. NdmC: 146.1 ng/uL
7. NdmD: 104.8ng/uL

2c: DpnI digest of vector PCRs

1.
15 uL vector 1
5 uL 10x NEB 4 Buffer
1 uL dpnI
29 uL H20
--
50 uL

2.
30 uL vector 2
5 uL 10x NEB 4 Buffer
1 uL dpnI
14 uL H20
--
50 uL

37 deg x 2 hrs

2e. Purification

Standard Qiagen PCR purification protocol. Elute in H20.


2F. Gibson cloning

pmols = weight(ng) x 1000 / (bp x 650 daltons)


Will use .05 pmol of vector, .1 pmol for each insert

Vec 1 (.05pmol) = need 68.57ng / (79.5ng/uL) = .86uL
Vec 2 (.05pmol) = need 68.57ng / (132.5ng/uL) = .52uL

NdmA1 (.1pmol) = need 71.5ng / (139.8ng/uL) = .51 uL
NdmA2 (.1pmol) = need 71.5ng / (144.5ng/uL) = .49 uL
NdmB (.1pmol) = need 73.4ng / (144.6ng/uL) = .50 uL
NdmC (.1pmol) = need 58.8ng / (146.1ng/uL) = .40 uL
NdmD (.1pmol) = need 120.ng / (104.8ng/uL) = 1.15 uL

rxns:

1.
.86 uL vec 1
.51 uL ndmA_1
.50 uL NdmB
.40 uL NdmC
1.15 uL NdmD
15 ul 1.33x Gibson Master Mix
1.58 uL H20
--
20 uL total

2.
.86 uL vec 1
15 uL 1.33x Gibson Master Mix
4.14 uL H20
--
20 uL total

3.
.52 uL Vec 2
.51 uL ndmA_1
.50 uL NdmB
.40 uL NdmC
1.15 uL NdmD
15 ul 1.33x Gibson Master Mix
1.94 uL H20
--
20 uL total

4.
.52 uL vec 1
15 uL 1.33x Gibson Master Mix
4.14 uL H20
--
20 uL total

50 deg thermocycler x 60 minutes

Run 5 uL each reaction (1-4)on .8% agarose gel + 3.5 ul Fermentas 1kb GeneRuler

2f.jpg


Desalted gibson reactions on .025uM Nitrocellulose membranes x 20 minutes

2g. Electroporation

Decided to not proceed with promoterless construct (2F3,4)

1 uL desalted gibson reaction (2F1,2) ->50 uL electrocompetent BW25113-GuaB -> 1mL SOC -> 1hr incubation @ 37 deg


2j. Selective growth in Caffeine/Theophylline

dilute 60 uL 2g transformations (1-2) -> 3mL Mineral M9 media + 34ug/mL chloramphenicol +/- 100mg Caffeine or Theophylline. Place on 30 deg shaker x 48 hrs

Results (growth):

Media 2g1 (+operon) 2g2 (vector only)
M9 - -
M9 + Caffeine - -
M9 + Theophylline + -


Result: construct enables growth (demethylation) on Theophylline. NdmC is not fuctional.

Streak 2j1 theophylline enriched culture onto LB-chloramphenicol plate for isolation of single colonies.

2K Plasmid Prep

Pick 2 colonies from theophylline enriched streak for plasmid prep. Prep 5mL culture.

2L Restriction Digest

Perform restriction digest to analyze insert size of plasmids

1 uL 2k1,2
1 uL 10x NEB 3
.5 uL NotI
6.5 uL H20
--
10 uL

37 deg x 1hr

Run 5 uL on .8% agarose gel

2L.jpg

Lanes:
1. 2k1
2. 2k2
3. GeneRuler 1kb ladder

Result: both clones show anticipated ~5kB band corresponding to the complete operon.

Retrieved from "http://2012.igem.org/Refactored_Decaffeination_Operon"