Overview of Bindley
Bioscience Center
Protein Production Lab:
experimental capabilities.
Bindley Biosceince Center, room 222
Phone 496-3102
http://www. ...? is coming
Larisa Avramova
Contact
Multiple samples?
Repeating assays?
Tired of manual pipetting?

We can help!
Running of experiment with many samples in parallel (high
throughput, HTP) requires the special labware: the 96 well plates.
Different types of plates exist – PCR, filtration, deepwell.
PCR plate
Standard
Deepwell plate
Filtration plate
That is why all instruments in the our lab have to be outfitted to handle
plates. And they are!
Equipment designed for High Throughput experiments:
1. Beckman HighThroughput Liquid Handling System “ BiomekF
Dual arm for 8-tip and 96–tip pipettors,
Filtration module,
Absorbance-plate reading module,
8 Modules for fixing 96-wells plate to the instrument
2 cooling-heating modules,
Waste disposal module.
2. MJResearch PCR Thermocycler
“DNA Engine DYAD”
(gradient available).
3. Sigma-Quiagen Centrifuge
4K15C with rotor for 96-well plates.
4. Amersham Biosciences Liquid Chromatography system.
AKTAexplorer100 ,
Autosampler A-905,
Frac-950 fraction collector
with rack for 96-well plate,
Midwest Scientific
Chromatography refrigerator.
5. Kuehner AG Cryo replicator System “Duetz”.
6. Ready-to-run electrophoresis systems
for 96 sample of DNA, Invitrogen,
Vertical and ready-to-run
PAGE-SDSsystems for 26, 52 and 96
sample of protein, Invitrogen, BioRad
7. Multichannel pipettors.
“Biohit”
“Matrix”
min 0.2ul
max 1200ul
Max 1250ul min 1ul
8. Thermomixer, shaker, heated/refrigerated incubator
with holders for microtiter plates.
Vortex
Benchtop Model Shaker-Incubator
“Innova 4230”
Rocking
platform
Orbital
shaker
Thermomixer
Applications
(including but not limited to)
High-throughput cloning:
Generation of PCR fragments.
Analysis by agarose gel.
Digestion with cloning enzymes.
Ligation of fragments.
Bacterial transformation and growth (plasmid propagation).
Isolation of plasmid DNA.
Verification of correct plasmid.
Screening for soluble protein:
Transformation (protein expression).
Screening of growth conditions.
Preparation of soluble/insoluble fraction and their analysis by PAGE.
Custom design of assays and experiments to suit your particular
high-throughput needs
Design of Constructs
•Different types of tag: His, MBP, GST, Trx,
•Different location of tag: N- or C-termini
•Different size: full length or truncated protein
•Different sources
Standard Steps for Cloning Experiment
 Target choosing
 Vector preparation


Isolation of plasmid vector
Digestion with cloning enzymes
 Insert preparation
 Primer design
 PCR fragment generation
 Digestion with cloning
enzymes
 Ligation
 Bacterial transformation and growth (plasmid propagation)
 Plasmid DNA isolation, verification of correct plasmid
 Bacterial transformation and growth (protein expression)
 Screening for soluble protein
 Purification of protein
Invitrogen,
Precast 1% agarose gel
PCR Amplification
~1500bp
~500bp
Bacterial transformation


Chemical
Heat-shock
Q-tray
Procedure for plasmid purification
96 x 1–1.25 ml E. coli bacterial culture in 96-Deepwell Culture Plate
Centrifuge Deepwell Culture Plate. Pour off or aspirate supernatants
Add Resuspension buffer.
Add Lysis buffer.
Add Neutralization buffer
Place Filter Plate A
on top of Filter
Plate DB
Pipette each lysate in the Culture Plate to Filter Plate A. Apply vacuum.
Add DNA Binding Buffer to Filter Plate DB (on top of Manifold).
Apply vacuum .
Add Washing buffer to Filter Plate DB. Apply vacuum.
Add Molecular Biology Grade Water to DNA binding matrix in Filter
Plate DB. Incubate.
Place Filter Plate
DB
on top of
Collection
Plate
Gradually apply vacuum to elute plasmid DNA into Collection Plate
Result: purified plasmid DNA in Collection Plate
Protein expression
LB media
autoinduction
18 C
25C
IPTG induction
37C
Low IPTG
High IPTG
18C
18C
25C
25C
37C
37C
Screening for Soluble Protein
Separate phases
by Filtration (Invitrogen)
MW,
kDa
110
80
56
35
30
25
Soluble fraction
Insoluble fraction
Purification of Protein
Scale up expression of successful constructs
or
 Purify protein from the soluble fraction with Ni
resin on the plate
or
 Purify protein from soluble
fraction using
AKTA autosampler

Efficiency of Steps
PCR: 100%


94 out of 96 – first round ( robotic)
96 out of 96 – after manual repeating of 2 samples
Correct plasmid: 96%


55 positive, 3 negative, 24 unclear, 14 no information – after screening
of 2 plates
92 positive, 4 no insert after screening of additional 2 plates
Expression of protein (1 set of conditions):

51 – expression of protein, 34 – expression in soluble fraction
Scale up of protein expression:

1 – soluble fraction
Finance
step
PCR
Agarose
gel
Clean up
PCR
Digestion
Clean up
digestion
Ligation
Transfor
mation
Plasmid
purificati
on
Plasmid
digestion
Agarose
gel
Transfor
mation
Cell
growth
Preparati
on
soluble
and
insoluble
fractions
SDSPAGE
Ni-plate
chemicals
Oligo 20c/base
~$7/oligo
Pfx polymerase
~$100/plate
dNTP
~$20/plate
Precast gel
$20/gel
Ultrafiltration
plate $33/plate
Enzymes
~$20/plate
Ultrafiltration
plate $33/plate
Ligase
~$100/plate
Plate
1PCR
Tips
3
accessories
Sealing – 1
PCR, 1 foil
1
1
1 foil
1
$24
1 standard
2-3
1 scotch
1
$46
1PCR
1
1foil
1
$29
1 standard
2-3
1 foil
1
$46
1PCR
2
1 foil
1
$113
1
1 foil
1
$20
4
4 breathable
4 foil
4
$90x4=360
1
1foil
4
$29x4=116
1
1 foil
4
$24x4=96
1
1 foil
1
$20
2
1 foil, 1
breathable
1-6
$13/plate
$13-78
1
2 foil
1-6
$75/plate
2
well
Qiagen Turbo kit
$182/plate
Eppendorf kit
~$70/plate
Enzymes
~$20/plate
Precast gel
$20/gel
48-
1PCR
2
48well
1 standard
1
deepwell
Invitrogen kit
~$70/plate
Total price
$120
$350700(oligo)
$75-450
$13/gel
8 gel /plate
~$65/plate
2 PCR
4
2 foil
1-6
2
2
1 foil
1
$130/plate
130-780
$75
$2300.00
(70 oligo, 4
plasmid
preps, 4
protein
$900.00
+
Price
of
oligo