Lab 7: Harvest Cells (EXTRA LAB)
Objective:
Your cells have been growing in liquid for 20 hours. Today we will archive your clones
(should you need to work with them in the future) and spin down our cells. The cells will
then be placed in the -80 oC freezer until next week. This will lyse the cells and prepare
them for our plasmid prep.
Pre-lab Exercise 7 & 8:
1. What does the term “plasmid prep” mean? (4 points)
2. What is the purpose of saving your plate cultures with glycerol at –80oC? (2
points)
3. What solution is used at the end of the procedure to dissolve the plasmid? (2
points)
4. What setting is used for the centrifugation of the plates? (2 points)
Supplies (per person unless otherwise stated):
 Bunsen burner
 Ethanol spray bottle
 Paper towels
 Kim wipes
 Flint
 Waste container
 Biowaste container (one large one for entire lab)
 Laboratory tape
 Ethanol resistant marker
 Stopwatch
 Round bottom 1ml sterile Nunc 96 well plate with lid (1 per student)
 Mylar plate sealers (1 per student)
 Multichannel pipette (40-200 l range, 1 set per 2 students)
 Pipette tips (2-200 l range) (7 boxes of 96 per student!!!)
 Multichannel pipette reservoir (4 per student)
 Eppendorf centrifuge with 96 well plate rotor at 4oC
 Vortex with 96 well plate adapter
Solutions:
 HAVE CLONE PLATES READY FOR EACH STUDENT
 Sterile glycerol
Plasmid Prep Protocol:
___ 1.
You will have a 96 well NUNC plate at your bench with 20uL of
glycerol in each well (Keep it sterile).
___ 2.
___ 3.
___ 4.
Transfer 100 l of your clones from the 96 well deep plate to this
new plate to archive your clones. MAKE SURE YOU LABEL
THE PLATE (not just the lid).
Seal the plate with mylar and give to me for archiving at –80oC.
Spin the remaining culture in the deep well plate for 15 minutes at
3300 rpm (we will need to go to the 3rd floor lab for this).
___ 5.
___ 6.
Invert the plate over the biohazerd waste to remove the
supernatant while retaining the bacterial pellet… ASK ME HOW!
Store plate at –80oC until next week.
Report Guidelines for Laboratory 7 & 8:
1. Did your clones grow in the presence of absence of ampicillin? Why? (4
points)
2. Was there any observable turbidity in your 96-well deep plate? (2 points)
3. Why did we centrifuge the plates? (2 points)
4. Why did we archive a portion of our clones? What chemical was added to
the plates intended for archiving? (3 points)
5. Are there any potential sources of contamination in the lab 7 & 8? (5
points)
6. Is there any white precipitation in your final plasmid plate? (2 points)
7. What do we assume is in the plasmid plate? (2 points)
8. If the goal is to re-amplify the 16S rRNA genes from the soil environment,
why can we not use the 8F and 1492R primers with the plasmid samples
as template? (5 points)
Lab 8: Plasmid Prep
Objective:
Last week, you cloned your community 16S rRNA genes using the Invitrogen TOPO TA
cloning kit. The clones you picked were then grown on solid followed by liquid media.
Today you will extract plasmid (perform a “plasmid prep”) from your clones.
Supplies (per person unless otherwise stated):
 Bunsen burner
 Ethanol spray bottle
 Paper towels
 Kim wipes
 Flint
 Waste container
 Biowaste container (one large one for entire lab)
 Laboratory tape
 Ethanol resistant marker
 Stopwatch
 Round bottom 1ml sterile Nunc 96 well plate with lid (2 per student)
 Mylar plate sealers (2 per student)
 Multichannel pipette (40-200 l range, 1 set per 2 students)
 Pipette tips (2-200 l range) (7 boxes of 96 per student!!!)
 Multichannel pipette reservoir (4 per student)
 Eppendorf centrifuge with 96 well plate rotor at 4oC
 Vortex with 96 well plate adapter
Solutions:
 HAVE CLONE PLATES READY FOR EACH STUDENT
 Sterile glycerol
 P1 solution
 P2 solution
 P3 solution
 Cold Isopropanol
 TE Buffer
___ 1.
___ 2.
___ 3.
Add 100 l of P1 solution to each well, place the plate on the
vortex and turn on for 2 minutes at setting 7.
Add 100 l of P1 solution to each well, place the plate on the
vortex and turn on for 1 minutes at setting 3.
Place the plate on ice for 5 minutes.
___ 4.
___ 5.
___ 6.
___ 7.
___ 8.
___ 9.
___ 10.
___ 11.
___ 12.
___ 13.
Add 100 l of P3 solution to each well, place the plate on the
vortex and turn on for 2 minutes at setting 7.
Place the plate on ice for 20 minutes.
Spin the lysed culture in the deep well plate for 10 minutes at
3300 rpm.
GENLTY remove 200 l of the supernant without disturbing the
cell debris pellet to a fresh 96 well sterile Nunc plate. Label this
new plate with your name and sample information.
Add 200 l of cold isopropanol to the Nunc plate.
Spin the lysed culture in the deep well plate for 10 minutes at
3600 rpm.
Invert the plate over the sink to remove the supernatant and dry
the plate at your bench for 30 minutes face down.
Add 40 l of TE buffer to each well to dissolve your plasmid.
Seal the plate with mylar sealer.
Place lid on the plate and store at 4oC.