Chm 382 Cloning 22006

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Cloning a DNA segment from bacteriophage lambda

Recombinant DNA transformed into bacterial cells

Preparation 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 bacteria

Plating of bacteria onto agar plates + ampicillin + X-gal

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Electrophoretic analysis of restriction digestsReceive agarose gel with ethidium bromide - done by Dr. Soukup

Load restriction digests on gel with size standardsExamine results

Agarose gel separates larger DNA molecules by size

Ethidium Bromide fluoresces under UV light

EB intercalates into DNA

Put gel on UV light source after electrophoresis

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

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Plasmid 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

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Plasmid characteristics

Ampicillin: antibiotic used to kill bacteria by interfering with synthesis of bacterial cell wall and leads

to lysis of bacteria

Ampicillin 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-resistantgenes

Today we will make one of these plasmids - ampicillin resistance gene codes for Beta-lactamase

(penicillinase) that inactivates (degrades) ampicillin

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Making 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)

CaCl2causes 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 C

Next day use small culture to seed large culture and grow to mid-log phase growth

Wash the cells with CaCl2

Incubate the cells at 4 C for 12 hours

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Making of competent cells

Bacterial growth in liquid media

Exponential growth occurs until no O2left

Measure cell growth by:

cell count (microscope)

cell mass (A600)

< 90 min

Doubling

time = 20 min

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Transformation 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 min

USE 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 CaCl2treatment (repairs its membrane)

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

USE STERILE TECHNIQUES!!!! MUST HAVE FLAME ON!!!

You will be spreading your bacteria onto the agar plate using a glass rod

Pipet culture onto plate and then spread using STERILE TECHNIQUES!!

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

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Controls:

E.coli-pUC18 negative control

Should only get blue colonies

E.coli-pUC18-satellite positive control

Should only get white colonies

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Plasmid 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 colored

X-gal converted to blue product by -galactosidase

pUC18 has a portion of Lac Z gene, remaining

portion 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 BLUE

If foreign DNA (bacteriophage DNA) inserted - complementation is destroyed and no active -gal, so colonies

WHITE

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Possible reasons for WHITE COLONIES???

RESTRICTION DIGESTS

RECIRCULARIZATION OF pUC18 during ligation with no foreign DNA inserted

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Safety

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!!

Documents

Chm 382 Cloning 22006