mystery plasmid b ??????
DESCRIPTION
Mystery Plasmid B ??????. Preliminary Work. 18 Possible Plasmids 11 Available Enzymes Too much work… Simplify: 3 Digestions. First Set of Digestions. AseI, BamHI Double Digestion DNA3 μ L (1000ng/ μ L) AseI.75 μ L BamHI.75 μ L BSA1.5 μ L Buffer 31.5 μ L H 2 07.5 μ L - PowerPoint PPT PresentationTRANSCRIPT
Mystery Plasmid B
??????
Preliminary Work
18 Possible Plasmids 11 Available EnzymesToo much work…
Simplify: 3 Digestions
First Set of Digestions
BglI Single Digestion DNA 3 μL
(1000ng/μL)
BglI 1 μL Buffer 3 1.5
μL H2O 9.5
μL
Total 15 μL
AseI, BamHI Double Digestion DNA 3 μL
(1000ng/μL)
AseI .75 μL
BamHI .75 μL
BSA 1.5 μL
Buffer 3 1.5 μL
H20 7.5 μL
Total 15 μL
AseI, NcoI Double Digestion DNA 3 μL
(1000ng/μL)
AseI .75 μL
NcoI1 .75 μL BSA 1.5
μL Buffer 3 1.5
μL H20 7.5
μL
Total 15 μL
All Buffers 100% Efficiency
2-Lo
g La
dder
Unc
ut P
lasm
idB
glI
Ase
I, B
amH
IA
seI,
Nco
I2-
Log
Ladd
er
First Gel
10 kb
3.0 kb
4.0 kb
2.0 kb
1.5 kb
1.2 kb1.0 kb
0.5 kb
0.1 kb
10 kb
3.0 kb
4.0 kb
2.0 kb
1.5 kb
1.2 kb
1.0 kb
0.5 kb
0.1 kb
2-Lo
g La
dder
Unc
ut P
lasm
idB
glI
Ase
I, B
amH
IA
seI,
Nco
I2-
Log
Ladd
er
All complete digestions
BglI:
Single Restriction Site
~3,500 – 4,500
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 Single Restriction Site Linearizes DNA Size of Plasmid: little greater than
4,000 bp
ONLY PLASMIDS WITH 1 BglI SitepGLO 5371 bppGPS3 4293 bppACYC177 3941 bppBAD-HisB 4092 bppET17b 3306 bp
Possible Couldn’t See Small BandpGPS1.1 4580 bp
234 bp
10 kb
3.0 kb
4.0 kb
2.0 kb
1.5 kb
1.2 kb
1.0 kb
0.5 kb
0.1 kb
2-Lo
g La
dder
Unc
ut P
lasm
idB
glI
AseI, BamHI 3 Restriction
Sites~3,500 - 2,500~1,500 – 1,000~200 – 100
pBAD-HisB 27401180 172
pEcoli-HN-GFPuv2998 1424
Bgl
I
Ase
I, B
amH
IA
seI,
Nco
I2-
Log
Ladd
er
10 kb
3.0 kb
4.0 kb
2.0 kb
1.5 kb
1.2 kb1.0 kb
0.5 kb
0.1 kb
Bgl
I
Ase
I, B
amH
IA
seI,
Nco
I2-
Log
Ladd
erpET11c
2922 1424 1142 125 59
pGEM-T-MC1-RFP2437 1235 59
AseI, NcoI 2 Restriction
Sites~ 3,500 – 2,500~ 1,500 – 1,200
pACYC17721831758
pBAD-HisB2818 1274
Bgl
IA
seI,
Bam
HI
Ase
I, N
coI
2-Lo
g La
dder
10 kb
3.0 kb
4.0 kb
2.0 kb
1.5 kb
1.2 kb1.0 kb
0.5 kb
0.1 kb
Bgl
I
Ase
I, N
coI
2-Lo
g La
dder
pGLO3633 1738
pGPS32424 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 pGEM-T-MC1-RFP
pET17b pET11c
pBAD-HisBGel labeled with calculated pBAD-HisB bands from restriction sites
Match the gel perfectly!
2-Lo
g La
dder
Unc
ut P
lasm
idB
glI
Ase
I, B
amH
IA
seI,
Nco
I2-
Log
Ladd
er
10 kb
3.0 kb
4.0 kb
2.0 kb
1.5 kb
1.2 kb1.0 kb
0.5 kb
0.1 kb
10 kb
3.0 kb
4.0 kb
2.0 kb
1.5 kb
1.2 kb
1.0 kb
0.5 kb
0.1 kb
2-Lo
g La
dder
Unc
ut P
lasm
idB
glI
Ase
I, B
amH
IA
seI,
Nco
I2-
Log
Ladd
er
2740
4092
1180
172
2818
1274
Extra Digestions:Chose 5 additional digestions to confidently identify plasmid
EcoRI, EcoRVDNA 3 μLBuffer 3 1.5 μLEcoRI .75 μLEcoRV .75 μLBSA 1.5 μLH20
7.5 μLTotal 15 μL
NdeIDNA 3
μLBuffer 2
1.5 μLNdeI 1
μLH20
9.5 μL
Total 15 μL
AseI, BglIIDNA 3
μLBuffer 3
1.5 μLAseI
.75 μLBglII
.75 μLH20
7.5 μL
Total 15 μL
NdeI, SphIDNA 3
μLBuffer 2
1.5 μLNdeI
.75 μLSphI
.75 μLH20
9 μL
Total 15 μL
PstIDNA 3
μLBuffer 3
1.5 μLPstI 1
μLBSA
1.5 μLH20
8 μL
Total 15 μL
All Buffers 100% Efficiency
pBAD-HisB
10 kb
3.0 kb
4.0 kb
2.0 kb
1.5 kb
1.2 kb1.0 kb
0.5 kb
0.1 kb
10 kb
3.0 kb
4.0 kb
2.0 kb
1.5 kb
1.2 kb
1.0 kb
0.5 kb
0.1 kb
2-Lo
g La
dder
Unc
ut P
lasm
idE
coR
I, E
coR
VN
deI
Ase
I, B
glII
2-Lo
g La
dder
Nde
I, S
phI
Pst
I
Final DigestionExpected pBAD-HisB
Bands
EcoRV, EcoRI3030 1062
NdeI2391 1701
AseI, BglII2925 1167
NdeI, SphI2391 1435 266
Pst4092
3030
1062
2391
1701
2925
1167
2391
1435
266
4092
My Mystery Plasmid is:
pBAD-HisB