Transcript Sample Mystery Plasmid Presentation

Mystery Plasmid B
??????
Preliminary Work
18 Possible Plasmids
 11 Available Enzymes
Too much work…
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Simplify: 3 Digestions
First Set of Digestions
BglI
Single Digestion
 DNA
3 μL
AseI, BamHI
Double Digestion
 DNA
3 μL
(1000ng/μL)
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BglI
Buffer 3
H2O
(1000ng/μL)
1 μL
1.5 μL
9.5 μL
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Total
AseI, NcoI
Double Digestion
 DNA
3 μL
15 μL
AseI
BamHI
BSA
Buffer 3
H20
Total
All Buffers 100% Efficiency
(1000ng/μL)
.75 μL
.75 μL
1.5 μL
1.5 μL
7.5 μL
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15 μL
Total
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AseI
NcoI1
BSA
Buffer 3
H20
.75 μL
.75 μL
1.5 μL
1.5 μL
7.5 μL
15 μL
All complete digestions
First Gel
BglI:
Single Restriction Site
~3,500 – 4,500
10 kb
10 kb
4.0 kb
4.0 kb
3.0 kb
3.0 kb
2.0 kb
2.0 kb
1.5 kb
1.5 kb
1.2 kb
1.0 kb
1.2 kb
1.0 kb
0.5 kb
0.5 kb
0.1 kb
0.1 kb
AseI, BamHI:
3 Restriction Sites
~3,500 - 2,500
~1,500 – 1,000
~200 – 100
AseI, NcoI:
2 Restriction Sites
~ 3,500 – 2,500
~ 1,500 – 1,200
BglI Single Digestion
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Single Restriction Site
Linearizes DNA
Size of Plasmid: little greater than
4,000 bp
10 kb
4.0 kb
ONLY PLASMIDS WITH 1 BglI Site
pGLO
5371 bp
pGPS3
4293 bp
pACYC177
3941 bp
pBAD-HisB
4092 bp
pET17b
3306 bp
Possible Couldn’t See Small Band
pGPS1.1
4580 bp
234 bp
3.0 kb
2.0 kb
1.5 kb
1.2 kb
1.0 kb
0.5 kb
0.1 kb
AseI, BamHI
3 Restriction Sites
~3,500 - 2,500
~1,500 – 1,000
~200 – 100
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10 kb
4.0 kb
3.0 kb
2.0 kb
1.5 kb
1.2 kb
1.0 kb
pBAD-HisB
2740
1180
172
pET11c
pEcoli-HN-GFPuv
2998
1424
pGEM-T-MC1-RFP
2437
1235
59
0.5 kb
0.1 kb
2922
1424
1142
125
59
AseI, NcoI
2 Restriction Sites
~ 3,500 – 2,500
~ 1,500 – 1,200

10 kb
4.0 kb
3.0 kb
2.0 kb
1.5 kb
1.2 kb
1.0 kb
pACYC177
2183
1758
pGLO
pBAD-HisB
2818
1274
pGPS3
3633
1738
0.5 kb
0.1 kb
2424
1869
All Three Digestions
BglI
AseI, BamHI
AseI, NcoI
pBAD-HisB
pBAD-HisB
pBAD-HisB
pGLO
pEcoli-HN-GFPuv
pGLO
pGPS3
pGPS3
pACYC177
pACYC177
pGPS1.1
pET17b
pGEM-T-MC1-RFP
pET11c
pBAD-HisB
Gel labeled with
calculated
pBAD-HisB
bands from
restriction sites
Match the gel
perfectly!
10 kb
10 kb
4092
4.0 kb
4.0 kb
3.0 kb
2740
2818
2.0 kb
2.0 kb
1.5 kb
3.0 kb
1180
1274
1.5 kb
1.2 kb
1.0 kb
1.2 kb
1.0 kb
0.5 kb
0.5 kb
172
0.1 kb
0.1 kb
Extra Digestions:
Chose 5 additional digestions to confidently identify plasmid
EcoRI, EcoRV
DNA
3 μL
Buffer 3 1.5 μL
EcoRI
.75 μL
EcoRV
.75 μL
BSA
1.5 μL
H20
7.5 μL
Total
15 μL
NdeI
DNA
Buffer 2
NdeI
H20
3 μL
1.5 μL
1 μL
9.5 μL
AseI, BglII
DNA
3 μL
Buffer 3 1.5 μL
AseI
.75 μL
BglII
.75 μL
H20
7.5 μL
Total
15 μL
Total
NdeI, SphI
DNA
3 μL
Buffer 2 1.5 μL
NdeI
.75 μL
SphI
.75 μL
H20
9 μL
PstI
DNA
Buffer 3
PstI
BSA
H20
3 μL
1.5 μL
1 μL
1.5 μL
8 μL
Total
Total
15 μL
15 μL
All Buffers 100% Efficiency
15 μL
pBAD-HisB
Final Digestion
Expected
pBAD-HisB Bands
EcoRV, EcoRI
3030
1062
10 kb
10 kb
4092
4.0 kb
3.0 kb
NdeI
2391
1701
1167
2925
2391
2391
2.0 kb
1435
1062
4.0 kb
3.0 kb
2.0 kb
1701
1.5 kb
1.2 kb
1.0 kb
AseI, BglII
2925
3030
1167
1.5 kb
1.2 kb
1.0 kb
0.5 kb
0.5 kb
266
NdeI, SphI
2391
1435
Pst
4092
266
0.1 kb
0.1 kb
My Mystery Plasmid is:
pBAD-HisB