Extract, purify, and quantify plasmid DNA from bacterial cells. This 2-day procedure involves lysing bacterial cells, removal of impurities and plasmid purification techniques. The resulting purified DNA product can be quantified, sequenced and stored for further analysis or use in cloning experiments.
Materials
GeneJET Plasmid Miniprep Kit
Resuspension Solution (Buffer 1)
Lysis Solution (Buffer 2)
Neutralization Solution (Buffer 3)
Wash Solution
RNase A
Elution Buffer (10 mM Tris-HCL, pH 8.5)
GeneJET Spin Columns
Collection Tubes (2 mL)
1.5 mL or 2 mL Eppendorf tubes
Molecular grade water (DNase/RNase-free deionized water)
96% Ethanol (only if you are opening a new kit)
Culturing media, generally Luria Broth (LB)
Antibiotic (if necessary)
Shaker
37°C incubator/room
Procedure
Set-up
Day 0 - Transformation
Transform E. coli with desired construct
Streak on proper plate Day 1 - Culture set up
Pick colonies from E. coli bacterial colony plates
Construct
# of colonies picked
[insert names of plates]
Start a 1 - 5 mL culture and grow at 37°C for 12 - 16 hrs (add necessary antibiotics)
Miniprep
GeneJET / QIAGEN Plasmid Miniprep Kit Protocol
transfer 1.5 mL of bacterial culture using pipette
into 1.5 mL microcentrifuge tube
into 15 mL Falcon tube
pellet bacteria by centrifuging for 3 minutes @ 8000 RPM, decant supernatant
can use pipette to remove supernatant if worried
resuspend in 250 µl resuspension Buffer P1
transfer to microcentrifuge tube if you did 1. b. with 15 mL Falcon tube
add 250 µl lysis Buffer P2 to tube + invert 4-6 times gently to mix
do not allow lysis reaction to occur for 5 minutes
for small-medium pellets, can add Buffer N3 immediately
for larger pellets, wait up to 5 minutes
add 350 µL neutralization Buffer N3 to tube + invert 4-6 times gently to mix
allow neutralization process to take 9 minutes
centrifuge lysate for 1 minutes @ max. speed 13,000 RPM
transfer the supernatant to a new microcentrifuge tube.
add 650 µL ethanol to the collected supernatant and pipette up and down to mix.
transfer 800 µL of the mix to a spin column and centrifuge Column for 1 minute, discard flow-through. Repeat until all of the mix has been spun through.
wash Column by adding 500 µL Buffer PB, centrifuge for 1 minute TWICE (13,000 RPM)
discard flow-through transfer to new tube, centrifuge for 2 minutes to dry
place Column in new 1.5 mL microcentrifuge tube
add 50 μL ULTRA PURE WATER, let stand for 1 min + centrifuge for 1 min
Buffer EB can be used as well, however sometimes the reagents in the buffer can inhibit/interfere with downstream usage
Pre-warm water to 50-60 °C for increased recovery
nanodrop final products
260/280 should be higher than 1.70
260/230 higher than 2.00
A260 (absorbance) should be between 0.1 - 1.0 for a reliable reading. Anything higher indicates that the machine is not properly calibrated or the sample concentration is too high (need to be diluted).
