Weigh out 3.625g of agar using the weighing boat and add it to the 250mL of water.
Weigh out 5.0g of LB Broth using the weighing boat and add it to the 250mL of water.
Screw lid on bottle loosely.
Place autoclave tape on the lid of the bottle.
Place tray with approximately 1cm of water in height into the autoclave.
Place 500mL bottle into autoclave with "liquid" selected and press "start".
After an hour, remove bottle from autoclave. Let bottle sit for approximately 30 minutes.
Add 370uL of chloramphenicol to the 250mL solution.
Pour solution into petri dishes, covering the bottom of the dish.
Place two stripes on each dish.
Let dishes sit overnight.
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2 - Dynabead Prep
Materials
B&W wash Buffer (2X and 1X)
Dynabeads
DNA solution
Magnets
PCR Tubes
Procedure
Pipette 50 microL of dynabeads in PCR tubes (3 of them)
Wash beads by pipetting in 200 microL of 1X B&W buffer
Vortex PCR tubes for about 30 seconds
Place a magnet at the bottom of the tube until a solid pellet of dynabeads forms at the bottom
Pipette out the supernatant leaving the pellet intact at the bottom
Repeat steps 2-5 two times (performed a total 3 times)
To each tube add 100 microL 2X buffer, 5 microL DNA (100 microM), and 95 microL of water
Incubate on stirring apparatus/agitator at room temperature for 15 minutes
Wash the beads again by repeating steps 2-5 three times
Add 105 microL 1X buffer to the tubes to resuspend the beads
Freeze PCR tubes at -20 degrees Celsius until they are needed
3 - Bacterial Transformation
For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
Place the mixture on ice for 30 minutes. Do not mix.
Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
Place on ice for 5 minutes. Do not mix.
Pipette 950 µl of room temperature SOC into the mixture.
Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
Warm selection plates to 37°C.
Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
Use a pipette tip to scrape off a colony and place the colony in 200 microL of ddH20
Take 21 microL of bacteria water and add it to a PCR tube
Add half a microliter of primer VR and another half microliter of primer VF2 to PCR tube
Add half a microliter of 10 mm dNTPs, 0.125 microliters of Taq Polymerase, and the appropriate amount of Standard Taq Polymerase Buffer (Usually 2.5 for a 25 microliter reaction)
Run in PCR machine with desired thermal cycling
Prepare a 1% agarose gel if one is not available
Prepare sample for gel electrophoresis using a proper proportion of 6X loading dye to sample
Load DNA ladder and sample into gel and run gel
Analyze gel using U.V. light and determine the approximate length of the amplified sequence
10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20
Running RO water or ddH20
Vacuum Oven
Vacuum Pump
Fume Hood
Petri dishes
Parafilm
Place untreated glass slides in a clean Petri dish
Submerge glass slides in 25% Ammonium Hydroxide overnight
Rinse the same glass slides under running RO water for 10 minutes
Rinse the slides with Anhydrous Ethanol
Immerse the slides in 1% 3-mercaptopropyl trimethoxysilane, 95% Ethanol, 16 mM acetic acid (pH 4.5) for 30 - 45 minutes
Rinse the same slides with 95% Ethanol, 16 mM acetic acid (pH 4.5)
Cure the glass slides in a vacuum oven at 150C for 2 hours
While slides are curing, suspend 1 - 40 uM of disulfide modified DNA in 150 uL of of 500 mM Sodium Bicarbonate Buffer (pH 9.0)
Remove cured slides from vacuum oven, and array disulfide modified DNA suspended in 500 mM Sodium Bicarbonate Buffer (pH 9.0) onto slides. Various arrays, ranging from 5 x 5 lattices using 2 uL dots to 1 large 50 uL dot were used
Place the slides in a moist environment, such as on a rack in a 30C water bath, overnight
Clean the slides using three washes of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20
Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl)
Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
Add 1 gel volume of isopropanol to the sample and mix.
Place a QIAquick spin column in a provided 2 ml collection tube.
To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
Discard flow-through and place QIAquick column back in the same collection tube.
(Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm).
Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed.
Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix
Add 350 µl Buffer N3 and invert the tube immediately but gently 4–6 times.
Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
Centrifuge for 30–60 s. Discard the flow-through.
(Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl
Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
If pH indicator has been added to Buffer PB, check that the color of the mixture is yellow.
Place a QIAquick spin column in a provided 2 ml collection tube.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube.
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min.