BIOTECH QUALITY & REGULATIONS TRAINING
WORKSHOP DOCUMENT WRITING/LAB ACTIVITIES
At Ivy Tech Community College, Bloomington, IN
Tuesday, December 13 to Friday, December 16, 2011
8:00 AM to 6:00 PM
Group 1 – pUC19 Plasmid Isolation (Mini Prep)
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Transfer overnight culture into a 1.5 ml microcentrifuge tube.
Collect cell pellet by spinning (20 sec. at 15,000 rpm) the tube in a microcentrifuge.
Aspirate off the supernatant and repeat the above steps again.
Resuspend the pellet in 200 µl of TE. Incubate on ice for 5 min.
Add 360 µl of fresh made alkaline solutions (CSH solution II: 0.2 M NaOH and 1% SDS) and mix
by inverting the tube 10 times. Incubate on ice for 5 min.
Add 300 µl of CSH solution III and mix immediately by inverting the tube 10 times. Incubate on
ice 5 min.
Centrifuge the tube for 5 min at 15,000 rpm to separate the soluble fraction from the insoluble
cellular debris. Transfer the supernatant into a new tube.
Add an equal volume of phenol: chloroform solution to the tube and mix by vortexing 10-30
seconds.
*** Be cautious with phenol: chloroform solution! It can burn your skin! Proteins, lipids and
chromosomal DNA end up at interphase.
Centrifuge the tube (max speed) for 2 min and transfer the aqueous upper layer to a new tube.
Precipitate DNA by adding 2 volumes of 95% ethanol and vortexing for a few seconds. Leave at
room temp for 2 min then centrifuge for 5 min at 13,000 rpm.
Aspirate the supernatant. Add 1 ml of 70% ethanol and recentrifuge as above.
Remove ethanol and dry the pellet using a speedvac for 3 min.
Resuspend the DNA pellet using TE.
Group 2 – pUC19 Plasmid DNA Digestion with Restriction Enzymes
Enzymes: PstI, ScaI from NEBioLabs; Buffer 3 optimal
Reaction [15 µl TOTAL reaction volume]:
1 µl (at X µg/ µl) of pUC19
0.5 µl (X units) of each enzyme
1.5 µl of 10 x buffer
X µl of water
Set-up: 4 Tubes:
DNA only
DNA + ScaI
DNA + PstI
DNA + ScaI + PstI
Incubate 1 hour at 37°C water bath
Group 3 – 10X TAE Preparation
*** Group 4 needs 1x TAE for gel
Recipe for 10x TAE buffer:
400 mM Tris (M.W.=121.14)
0.2 M Acetic acid (the concentration of the Acetic acid stock we have is 17.4 M)
10 mM EDTA pH8 (M.W. = 372.24)
Group 4 – Agarose Gel Preparation
Gel prep:
Buffer: 650 ml of 1x TAE
70 ml of 0.8% LE agarose gel
10 well comb
Group 5 – Electrophoretic Separation of Digested pUC19 Plasmid Fragments
Sample prep:
Add 3 µl of 6 X loading dye to 15 µl of each sample (18 µl TOTAL volume)
Sample loading:
Load 7 µl of λ/Hind III marker to the first well.
Load 18 µl of each sample with loading dye to the next 4 wells
Run gel 1 hour at 100V
Ethidium Bromide Staining:
Mix 5 µl EtBr (10 mg/ml) with 200 ml of nanopure water
Place the gel in the EtBr solution in a plastic container and shake the container for 30 min at 60
rpm.
Group 6 – Analysis of pUC19 Plasmid Fragments
Use Kodak picture document system for gel box for picture/ analysis
pUC19 plasmid map:
Lab Activity Scenario:
XYZ Company makes a biotech product: pUC19
Workflow below simulates downstream processing and QC check of
product
Need to draft work instruction documents to describe how to perform each
task, as well as accompanying batch record
Grow up
E. coli / pUC19
culture
Group 1:
Isolate
pUC19 plasmid
Group 3:
Prepare
10x TAE buffer
Group 2:
Digest
pUC19 plasmid
DNA
Group 4:
Prepare
0.8% agarose gel
Group 5:
Electrophoretically
separate
pUC19 plasmid
DNA fragments
Group 6:
Analyze
pUC19 plasmid
DNA fragments