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PCR
For 50 µL reaction:
Mixed 25 µL of Phusion polymerase master mix + 21.5 µL milli-q water + 1 µL template +
2.5 µL primer solution
o Primer solution was created by mixing 1 µL of each stock primer and 8 µL of milli-q
water
Gel Electrophoresis
Created 1% gel solution by adding 50 mL 1x TAE buffer to 0.5 g agarose and microwaving
for 90 s
Added 5 µL SYBR safe dye and poured solution into mold. Allowed to set for about 20
minutes
Loaded gel with 12 µL of 1 kB ladder
Mixed 3 µL of 6x loading dye and 15 µL of PCR reaction
o Loaded 15 µL
Ran gel at 100 V for 30 minutes
Wearing goggles and lab coat, visualized gel using UV illuminator, covering gel with shield
DNA Purification
Followed ThermoScientific kit using “PCR cleanup” directions with these modifications:
o Used 20 µL milli-q water instead of elution buffer
E. coli Transformation
Obtained cells from freezer and thawed on ice
Added DNA and mixed by flicking. Incubated on ice for 5 minutes
o 1-2 µL for plasmid DNA
o 10 µL for cloning
Heat shocked cells for 30 s in 42°C water bath
Incubated on ice for 2 minutes
Transferred cells to 1 mL of LB media in a 15 mL culture tube. Place in 37°C incubator (with
shaking) for 1 hour
o Plates were removed from fridge and left at RT about 30 minutes before plating
Plated cells using a spreader (created by holding a Pasteur pipette over the flame and
allowing to bend twice)
o 20 µL for standard transformation
o 100 µL for cloning
Plates were placed in 37°C incubator (w/o shaking) for 12-14 hours
Mini-Prep
Followed instruction from ThermoScientific kit with these notes:
o Cells were pelleted by spinning down culture tubes at 5000 x g for 10 minutes
o 500 µL of resuspension buffer was added to each pellet. 290 µL aliquots were
transferred into two separate tubes per culture
o Collected DNA by adding 35 µL of milli-q water in column
Compound Kd (µM)
3-Br-4-HB 170 ± 30
2BP 150 ± 12
4-Br-3-HBA 85 ± 6
4-Br-3-HPA 5.3 ± 0.3
2BP 0.21
4-Br-3-HBA 0.874 ± 0.027
4-Br-3-HPA 2.23 ± 0.026