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