Cloning a DNA segment from bacteriophage lambda

Recombinant DNA transformed into bacterial cellsPreparation of X-gal plates - by Dr. Soukup before lab Preparation of competent cells - by Dr. Soukup before lab

DURING LAB:Electrophoretic analysis of restriction digests

Transformation of recombinant plasmid into bacteriaPlating of bacteria onto agar plates + ampicillin + X-gal

Cloning a DNA segment from bacteriophage lambda

Electrophoretic analysis of restriction digestsReceive agarose gel with ethidium bromide - done by Dr. SoukupLoad restriction digests on gel with size standardsExamine results

Agarose gel separates larger DNA molecules by sizeEthidium Bromide fluoresces under UV lightEB intercalates into DNAPut gel on UV light source after electrophoresis

Cloning a DNA segment from bacteriophage lambda

Recombinant DNA transformed into bacterial cells

Transformation of recombinant plasmid into bacteria (cells that take up plasmid are “transformed”)

Cloning a DNA segment from bacteriophage lambda

Recombinant DNA transformed into bacterial cellsPlasmid characteristics - small circular double-stranded DNA, usually not necessary for survival BUT can carry genes that confer resistance to antibiotics or allow survival in certain environments

Cloning a DNA segment from bacteriophage lambda

Recombinant DNA transformed into bacterial cellsPlasmid characteristics

Ampicillin: antibiotic used to kill bacteria by interfering with synthesis of bacterial cell wall and leads to lysis of bacteriaAmpicillin is a broad-spectrum semi-synthetic penicillin that will kill gram-negative and gram-positive bacteria, includes E.coli and Salmonella

E.coli in nature can become resistant to ampicillin by taking up plasmids that contain Amp-resistant genes

Today we will make one of these plasmids - ampicillin resistance gene codes for Beta-lactamase (penicillinase) that inactivates (degrades) ampicillin

Cloning a DNA segment from bacteriophage lambda

Recombinant DNA transformed into bacterial cellsMaking of competent cells

Treat bacterial cells (E. coli strain DH5) with CaCl2, which will make them COMPETENT to take up plasmid DNA (plasmid DNA will enter the cell) CaCl2 causes small holes to form in the cell membrane that DNA can then traverse through

We have pre-made competent cells

LABORATORY PROCESS TO MAKE COMPETENT CELLS:Grow small culture of bacterial from a single colony overnight at 37 ˚CNext day use small culture to seed large culture and grow to mid-log phase growthWash the cells with CaCl2

Incubate the cells at 4 ˚C for 12 hours

Cloning a DNA segment from bacteriophage lambda

Recombinant DNA transformed into bacterial cellsMaking of competent cells

Bacterial growth in liquid mediaExponential growth occurs until no O2 leftMeasure cell growth by:

cell count (microscope)cell mass (A600)

< 90 min

Doubling time = 20 min

Cloning a DNA segment from bacteriophage lambda

Recombinant DNA transformed into bacterial cellsTransformation procedure

1. Cells + plasmid DNA - incubate on ice for 20 min (cells starting to take up plasmid)DO NOT VORTEX OR ROUGHLY FLICK TUBE WITH CELLS - THEY ARE VERY FRAGILE

2. Transfer tube to 37 ˚C for 5 min (heat shock - causes faster uptake of plasmid)

3. Add nutrient broth (media) without ampicillin and incubate at 37 ˚C for 45 minUSE STERILE TECHNIQUES!!!! MUST HAVE FLAME ON!!!During this 45 min the plasmid has time to start expressing the amp-resistance gene and the bacteria cell recovers from CaCl2 treatment (repairs its membrane)

Cloning a DNA segment from bacteriophage lambda

Recombinant DNA transformed into bacterial cellsTransformation procedure

4. Plate cells onto agar plates + ampicillin + X-galUSE STERILE TECHNIQUES!!!! MUST HAVE FLAME ON!!!

You will be spreading your bacteria onto the agar plate using a glass rodPipet culture onto plate and then spread using STERILE TECHNIQUES!!

Dr. Soukup will demonstrate how to produce single colonies of control plasmids

Cloning a DNA segment from bacteriophage lambdaRecombinant DNA transformed into bacterial cellsTransformation procedure

4. Plate cells onto agar plates + ampicillin + X-gal

Controls:E.coli-pUC18 “negative control”Should only get blue colonies

E.coli-pUC18-satellite “positive control”Should only get white colonies

Cloning a DNA segment from bacteriophage lambda

Recombinant DNA transformed into bacterial cellsTransformation procedure

4. Plate cells onto agar plates + ampicillin + X-galPlasmid also has Lac Z gene which codes for -galactosidase (hydrolyzes lactose, other -galactosides and X-gal)

X-gal is 5-bromo-4-chloro-3-indolyl-D galactoside - chromogenic substrate because its product is coloredX-gal converted to blue product by -galactosidase

pUC18 has a portion of Lac Z gene, remainingportion is encoded by E.coli strain

SO when cells transformed with pUC18

Complementation occurs and cells make active -gal

Active -gal causes blue colonies to be produced on agar with X-gal

CAN DETERMINE WHICH PLASMIDS HAVE FOREIGN DNA Polylinker is inserted in Lac Z gene - if no foreign DNA inserted then -gal made and colonies BLUEIf foreign DNA (bacteriophage DNA) inserted - complementation is destroyed and no active -gal, so colonies WHITE

Cloning a DNA segment from bacteriophage lambdaRecombinant DNA transformed into bacterial cellsTransformation procedure

4. Plate cells onto agar plates + ampicillin + X-galPossible reasons for WHITE COLONIES???

RESTRICTION DIGESTSRECIRCULARIZATION OF pUC18 during ligation with no foreign DNA inserted

Cloning a DNA segment from bacteriophage lambdaRecombinant DNA transformed into bacterial cellsSafety

WASH YOUR HANDS WITH SOAP!!!!

DISINFECT LAB BENCH WITH BLEACH OR ETHANOL SOLUTION

IF YOU SPILL BACTERIA TELL DR. SOUKUP

LIMIT EXPOSE OF BACTERIA TO AIR

PLACE ALL BACTERIAL WASTE IN RED BIOHAZARD BAGS

WEAR GLOVES!!