BIO 208NUCLEIC ACID

METHODS

Stephanie Schumaker

ObjectiveAlpha Amylase

Bacillus licheniformis

amyE gene

Escherichia coli

amyE gene•Part of the chromosome in

many strains of Bacillus•Circular with a size of about

4000 kilobases•Gene is about 1.6 kilobases

Procedure

Get the DNAPCR and produce biotin-labeled probeSouthern BlotCloning the amyE geneVerify and MapCheck Enzyme Activity of Clones

DNA isolation• Centrifugation• Vortex • Glass beads• Reagents

– Ethanol– Phenol:chloroform:

isoamyl alcohol– Sodium acetate– TE

Quantitation of DNA

DNA A260 nm Proteins A280

nm 1.8 to 2.0 ratio

is pure DNA

ul of diluted sample DNA Sample A260 nm (DNA) A280 nm (Proteins) Ratio10 A 0.054 0.024 2.21110 B 0.025 0.015 1.67310 AW 0.021 0.013 1.63210 BW 0.006 0.003 2.032

QUANTITATION of DNA

ul of diluted sample DNA Sample A260 nm (DNA) A280 nm (Proteins) Ratio10 A+B 0.140 0.059 2.38810 B Wash 0.011 0.001 7.12610 AW 1st exp 0 0 0.00010 B 1st exp 0.017 0.009 1.784

QUANTITATION of DNA

CONCENTRATION 260nm x 50ug/ml x DF

.054 x 50ug/ml x 100 = 270ug/ml

270ug/ml / 1000ul = .27ug/ul

About every ul has ¼ ug 4ul = 1ug4ul = 1ug

.7ug/ul and assumed it to be .5ug/ul due to RNA

About every ul has ½ ug2ul = 1ug2ul = 1ug

A reading of 1 @ 260nm =50ug/ml of double stranded DNA

Restriction Enzyme Cleavage

• Enzymatic digestion of the DNA cuts it into fragments of interest

• 3 types of enzymes were used

– EcoR I– Hind III– Cla I

Gels 1 and 3

Empty Marker Sample A 10-1

Sample B 10-1

Sample AW 10-1

Sample A 10-2

Sample B 10-2

Sample AW 10-2

Empty Empty Uncut DNA Hind III Marker EcoR I Cla I Empty

Gel 2

Inconclusive

PCR

•Denature DNA•Annealing of

primers• Synthesis

PCR Gels 1 and 2

Empty - DNA 15ul control 5ul control Experimental Marker Empty Empty

Empty Empty Empty Marker Empty Experimental Empty Empty

PCR and Biotin labeled probe

NonradioactiveChemically bonds to

base pair of a nucleotide

Taq will incorporate molecules of BIO-

deoxycytidine during PCR

Southern Blot

• Restriction enzyme cleavage

• Denaturation and transfer of DNA to a membrane

• Southern Hybridization and detection

Detection

Results were inconclusive

Cloning the amyE gene in 5 easy steps

1) Enzyme restriction digest of DNA sample

2) Enzyme restriction digest of DNA plasmid vector

3) Ligation of DNA sample products and plasmid vector

4) Transformation of E. coli with the ligation products

5) Growth on agar plates with selection for antibiotic resistance

Steps 1-3

Plasmid pRL498

Verify and Map

• PCR is used to verify positive clones that yield the 433bp product

• Make sure the correct fragment was inserted into the vector using enzyme cleavage

• Map the 6.3kb amyE plasmid DNA by cleavage and gel electrophoresis

Check for enzyme activity

quantitative alpha amylase assay break down starch into maltose use maltose standard curve to

determine amount producedMaltose Standards

y = 0.2969x + 0.0229

R2 = 0.9875

00.10.20.30.40.50.60.7

0 0.5 1 1.5 2 2.5

toal mass (mg) maltose

A 5

40 n

m