Lab 22 Goals and Objectives:

EDVOKIT#300: Blue/White Cloning of a DNA FragmentCalculate transformation efficienciesCompare control efficiency to cloned gene (white colony) efficiency

*Control = transformation efficiency of cells to take up intact plasmid*All ligation colonies = transformation efficiency + ligation efficiency*White ligation colonies = transformation efficiency plus efficiency of

ligation of desired gene into plasmid (cloning efficiency)Exercise 67 and Handout: PCR

**Five (5) groups possible! Same group for PCR and EDVOKIT #124Set up PCR reactions (supplement pg 89-90) and place in thermocycler

EDVOKIT#124: DNA-based Screening for SmallpoxPrepare DNA agarose gels, 2 per group (pg 7-9):

1 practice gel with three combs1 experimental gel with one comb

Wrap gels and store until next lab***Next lab gels will need to run for 1.5hrs so we will load our PCR

samples and begin running the gel BEFORE lecture!

count colonies to calculate efficiency: colonies per µg of DNA

transformation efficiency

all colonies = ligation efficiency

white colonies = cloning efficiency

Control Ligation Control Ligation

Control LigationLigationControl

Polymerase Chain Reaction (PCR)

Method for amplifying/copying DNA-can turn one copy into 1 X 109 copies in ~3hr-generate large quantities for study:

1. determine crime suspect from minute traces of body fluids/tissues

2. detect infectious disease3. gene cloning (to extract gene from source)

Minimum requirements:1. Template DNA (source you want to copy)2. DNA polymerase (enzyme to synthesize new DNA)3. Primers (ssDNA fragments, complementary to ends of

target DNA sequence)4. Free nucleotides (ATP, CTP, TTP, GTP)

DNA Synthesis in a tube (PCR)1. Double stranded DNA template must

be separated2. DNA primers base pair to ends of

single stranded target sequence3. DNA polymerase adds nucleotides to

the 3’ end of the primers by complementary base pairing free nucleotides to the template strand

4. Repeat steps 1-3: each new copy generated can serve as a template in the next round of replication

*1 DNA molecule X 30 rounds of replication = 1.1 X 109 molecules(1073741824)

Fig.67.1

“Round of Replication” or cycle conditions (supp. pg.90)-in living cells (in vivo), enzymes and stabilizing proteins

assist the DNA polymerase in the synthesis process-in a tube (in vitro), temperature is varied to facilitate the

necessary molecular events in the absence of lifeHot Start - “melt” all the molecules and reagents (unstick,

unclump) and mix them together, done once at the beginning95°C, 30sec = denaturation: separate the double stranded

DNA template (break H-bonds)50-60°C, 30sec = annealing: allows primers to complementary

base pair with target sequence on template DNA, temp varied depending on %GC content

72°C, 1min/kb DNA = extension: optimal temp for DNA polymerase to synthesize new DNA molecules from ends of primers

Repeat cycle 20-30 times

Fig.67.1

Perform in a thermocycler

Use heat stable DNA polymerase: Taq

Lab 22 Goals and Objectives:

EDVOKIT#300: Blue/White Cloning of a DNA FragmentCalculate transformation efficienciesCompare control efficiency to cloned gene (white colony) efficiency

*Control = transformation efficiency of cells to take up intact plasmid*All ligation colonies = transformation efficiency + ligation efficiency*White ligation colonies = transformation efficiency plus efficiency of

ligation of desired gene into plasmid (cloning efficiency)Exercise 67 and Handout: PCR

**Five (5) groups possible! Same group for PCR and EDVOKIT #124Set up PCR reactions (supplement pg 89-90) and place in thermocycler

EDVOKIT#124: DNA-based Screening for SmallpoxPrepare DNA agarose gels, 2 per group (pg 7-9):

1 practice gel with three combs1 experimental gel with one comb

Wrap gels and store until next lab***Next lab gels will need to run for 1.5hrs so we will load our PCR

samples and begin running the gel BEFORE lecture!