Transcript June2014_BioLink_FasterBetterBiotech

Faster, Better Biotech:
Mini-Preps
Ellyn Daugherty
[email protected]
www.BiotechEd.com
Simon Holdaway
The Loomis Chaffee School
[email protected]
The rAmylase Project
Model of rDNA/protein Business
< Chapters 1-5 Basic SLOP
< Lab 5f Amylase PAGE (revised)
< Lab 6e Amylase Producing Bacteria
< Lab 6c Amylase Activity Assay (revised)
< Lab 6d Amylase ELISA and W Blot
< Labs 7a/7b/7f/7g Amy Spectrophotometry
< Lab 8b Restriction Digestion
< Lab 4j/8b/8g DNA Gel Electrophoresis
< Lab 8c pAmy Transformation of E. coli
< Lab 9c/9d Amy Ion-Ex Chromatography
< Lab 8g Plasmid DNA Isolation
< Lab 13i Amylase Gene PCR
The goal of The rAmylase Project is to
• Learn how to assay for a protein of commercial interest (amylase)
• Transform cells to produce that protein (using pAmylase and C2523 cells)
• Scale-up cells to volumes where amylase can be purified and assayed
Absorbance (a.u)
Ion Exchange Chromatography
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
-0.05 0
5
Fractions (#)
10
Find a product of interest (amylase) and make sure to
have assays to show it’s presence, activity, &
concentration
Amylase is an enzyme that
catalyzes starch digestion.
It is used commercially in
several ways including:
1) Remove starch in products
2) Produce sugar from starch
3) Processing of cellulostic
biofuel
The rAmylase Project
Model of rDNA/protein Business
< Chapters 1-5 Basic SLOP
< Lab 5f Amylase PAGE (revised)
< Lab 6e Amylase Producing Bacteria
< Lab 6c Amylase Activity Assay (revised)
< Lab 6d Amylase ELISA and W Blot
< Labs 7a/7b/7f/7g Amy Spectrophotometry
< Lab 8b Restriction Digestion
< Lab 4j/8b/8g DNA Gel Electrophoresis
< Lab 8c pAmy Transformation of E. coli
< Lab 9c/9d Amy Ion-Ex Chromatography
< Lab 8g Plasmid DNA Isolation
< Lab 13i Amylase Gene PCR
Faster, Better Plasmid Isolation
Lab 8g Zippy TM Plasmid Miniprep Kit (Zymo Research Corp #D4036)
It is often necessary to extract the
transforming plasmid from the transformed
cells. This is called a preparation (called
“prep,” for short).
When the amount of cells and cell culture
is small, the amount of plasmid recovered
from the cells is relatively small.
A miniprep is a plasmid isolation that yields
about 20 to 30 μg of DNA
(usually 20 μg in a 50 μL sample).
The “old mini-prep way” (Alkaline lysis)
requires cell pelleting and lots of nasty
buffers.
But now we have…
Zippy TM Plasmid
Miniprep Kit
Faster, Better Plasmid Isolation
Zippy TM Plasmid
Miniprep Kit Pre-lab prep
Grow 5 mL of overnight
transformed cell culture.
In six 50-mL tubes with 12 mL
sterile LB Broth, add 12µL of
1000X amp and 100 µL of
overnight culture.
Grow 18 hours at 37°C
Use 60 µL of this culture in each
mini-prep
Amylase
producing DH5alpha E. coli cells
Faster, Better Plasmid Isolation
Zippy TM Plasmid
Miniprep Kit
The fastest, easiest miniprep
available for purifying
transfection quality plasmid
DNA.
Pellet-free procedure omits
conventional cell pelleting and
re-suspension steps.
DNA quality appropriate for
cloning, sequencing, and
transfection.
Zyppy Protocol: The following procedure is performed at room temperature.
1.Add 600 µL of bacterial culture grown in LB medium to a 1.5 mL microcentrifuge tube.
2. Add 100 µL of 7X Lysis Buffer (Blue) and mix by inverting the tube 4-6 times. Proceed to
step 3 within 2 minutes.
After addition of 7X Lysis Buffer the solution should change from opaque to clear blue,
indicating complete lysis.
3. Add 350 μL of cold Neutralization Buffer (Yellow) and mix thoroughly.
The sample will turn yellow when the neutralization is complete and a yellowish precipitate will
form. Invert the sample an additional 2-3 times to ensure complete neutralization.
4. Centrifuge at 11,000 – 16,000 xg for 2-4 minutes.
5. Transfer the supernatant (~900 μL) into the provded Zymo-Spin™ IIN column. Avoid
disturbing the cell debris pellet.
6. Place the column into a Collection Tube and centrifuge for 15 seconds.
7. Discard the flow-through and place the column back into the same Collection Tube.
8. Add 200 μL of Endo-Wash Buffer to the column. Centrifuge for 30 seconds. It is not
necessary to empty the collection tube.
9. Add 400 µL of Zyppy™ Wash Buffer to the column. Centrifuge for 1 minute.
10. Transfer the column into a clean 1.5 mL microcentrifuge tube then add 30 µL of Zyppy™
Elution Buffer 2 directly to the column matrix and let stand for one minute at room
temperature.
11. Centrifuge for 30 seconds to elute the plasmid DNA
Faster Better Biotech: Plasmid Yield
How do you check to see if you got plasmid?
Old way…waste the small sample in restriction digest or UV spec.
Now…Use a G-Biosciences Nucleic dotMetric™ Assay kit (Geno
Technology Inc) to quantify the amount of DNA in the plasmid sample. Take
a 1 μL sample and blot it on the test strip.
Then using their DNA nucleic acid indicator and washes, stain the DNA
sample proportional to the mass present.
G-Biosciences Nucleic dotMetric Assay
1 µl Assay For Estimation of DNA Concentration (< 1 µg/µL)
1. Dilute samples with Nucleic Acid Dilution Buffer
(1:1 to 1:10 fold)
2. Apply 1-5 µl of each dilution onto the NUCLEIC Test Strip
(remove the protective cover 1st)
3. Developed with about 500 µL of NUCLEIC Dye (cover all
dots) about 1-2 minutes. Remove dye and wash/dunk in
dH20
4. Compare the color density of the nucleic acid spots to the
NUCLEIC standard strip.
No spectrophotometers or cuvettes required.
Faster Better Biotech:
DNA Band Visualization
1. Run samples that contain
µg amounts of (pAmy) DNA
on agarose gels
2. Can run make TAE agarose
gels (slow way) or use LB
buffer to make and run
gels….faster, better way.
Faster, Better Biotech:
DNA (Agarose) Gels
Gels made with
TAE Buffer
A tris acetate EDTA or tris borate
EDTA gel tolerates only 110 volts,
takes three times as long to run,
and results in fuzzy DNA patterns.
Tris also produces loads of
unwanted heat - heat that limits the
voltage that can be used.
•Slow gel runs of 45-60 min
•Steams up gel box
Gels made with
Lithium Borate (LB) Buffer
http://www.fasterbettermedia.com
Lithium borate conducts electricity
better than TRIS so you can turn up
the voltage and current on agarose
gels :
• Fast gel run times (8-15 min)
• Same protocol as TAE gels just
substitute 1X LB buffer for
1X TAE
• Purchase as 10X or 20X
concentrate
pAmy Visualization
Use 1X LB buffer/0.8% agarose gel with 2-6 well combs.
Have only 1 cm of running buffer over gel.
1. Add 2 µL of EZ-Vision Loading Dye to 10 µL of sample.
2. Load all 12 µL of each sample to a well. Have at least
one lane of DNA standard markers (Record what is
loaded in each well.
3. Run gel for 10-15 min at 300 V (or as close as you can
get)
4. Transfer gel to UV light box and visualize. Photograph.
Uncut pAmylase plasmid is about 7,000 bp.
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