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


Cloning a DNA segment from bacteriophage lambda
Electrophoretic analysis of restriction digests
Receive agarose gel with ethidium bromide - done by Dr. Soukup
Load restriction digests on gel with size standards
Examine 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

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 cells
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
Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial cells
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-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 cells
Making of competent cells

Treat bacterial cells (E. coli strain DH5) with CaCl
2
, which will make them COMPETENT to take
up plasmid DNA (plasmid DNA will enter the cell)
CaCl
2
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 C
Next day use small culture to seed large culture and grow to mid-log phase growth
Wash the cells with CaCl
2
Incubate the cells at 4 C for 12 hours

Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial cells
Making of competent cells

Bacterial growth in liquid media
Exponential growth occurs until no O
2
left
Measure cell growth by:
cell count (microscope)
cell mass (A
600
)

< 90 min
Doubling
time = 20 min
Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial cells
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 CaCl
2
treatment (repairs its membrane)
Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial cells
Transformation procedure

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
Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial cells
Transformation 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 cells
Transformation procedure

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

Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial cells
Transformation procedure

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

RESTRICTION DIGESTS
RECIRCULARIZATION OF pUC18 during ligation with no foreign DNA inserted
Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial cells
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!!