Team:OU Norman/Project/Protocols

Antibiotic (Tetracyclineb) Stock

Stock Solution

Working Concentrations

Physical Data Table

PROCEDURE FOR PREPARATION

• Obtain (3) 15 mL test tubes
• Obtain (1) 50 mL test tube
• Obtain 0.250 g of tetracycline
• Place in the 50 mL test tube
• Add EtOH to the 50 mL mark
• Shake to dissolve completely
• Use 100% EtOH
• Because the tetracycline did not dissolve, we removed 15 mL of the solution and added 15 mL of water to catalyze the reaction
• Next time we will use 35 mL of EtOH and 15 mL of water
• Pour 15 mL of the solution into each of the 15 mL test tubes
• Mark the test tubes in blue to identify tetracycline

PROCEDURE FOR BROTH AND PLATES

• Add 500 μL of the tetracycline antibiotic twice to the broth using an Eppendorf pipette
• Make sure the broth is cool before adding the antibiotic
• Pour the broth into 25 plates
• Allow the plates to cool before stacking them
• While the plates are cooling, place a second broth container on the spin plate
• Temperature = 150°C
• Rotation = 1000
• Once the plates have cooled mark them in blue (to identify as tetracycline), put them back in the sleeve, and store in the 5th floor refrigerator

Ligation of ClosOri, m1sR, and psB1K3

PROCEDURE

• Obtain (2) 1.5 mL centrifuge tubes
• In one of the centrifuge tubes, add:
• 20 μL of heat killed digest of ClosOri
• 20 μL of heat killed digest of psB1K3
• 1 μL of T4 ligase
• 5 μL of 10x T4 ligase buffer
• 4 μL of nuclease free PCR H2O
• In the second centrifuge tube, add:
• 20 μL of heat killed digest of m1sR
• 20 μL of heat killed digest of psB1K3
• 1 μL of T4 ligase
• 5 μL of 10x T4 ligase buffer
• 4 μL of nuclease free PCR H2O

Restriction Digest of ClosOri and m1sR

Conversions
ClosOri: 500 ng x 1 μL/ 210.2 ng = 2.38 μL
m1sR: 500 ng x 1 μL/ 470.6 ng = 1.05 μL
Backbone (psB1K3): 500 ng x 1 μL/ 175.2 ng = 2.86 μL
Physical Data Table

PROCEDURE

• Cross link water for 30 seconds
• Use DI water
• Take cap off of DI water when in spectrolinker
• Add appropriate amount of water for each of the four centrifuges (1.5 mL)
• Remember to keep the caps of the centrifuges on until they are in action
• Stick the pipette all the way down to the bottom of the centrifuge to ensure complete utilization of water
• Add buffer
• Add appropriate amounts of DNA
• Add restriction enzymes
• Spin the samples to briefly collect all of the mixture
• Incubate the restriction digests at 80°C for 15 minutes
• Use a thermal cycler with a heated lid
• Cool on the bench for ~5 minutes

Rescue of BBa K542003

DNA Sample = 2M on plate 1
Plasmid = pSC1C3

pBAD Regulated TetR
Production of TetR is regulated by the pBAD promoter. TetR is an inhibitor of the constitutively “on” promoter pTet-BBa_R0040. Therefore, this part may be used in conjunction with the pTet promoter of an “inverter”; pTet will be “turned off” in the presence of arabinose.
PROCEDURE

• Turn on heat bath to 42°C
• Obtain 10 μL of nuclease free PCR water
• Mix into cell 2 M of the titer plate
• Set for 5 minutes
• Put the mixture into the PCR tube

Nanodrop

• Obtain 2 μL of DI water
• Clean the lens with a kim wipe
• Drop the 2 μL of DI water on the aperature of the nanodrop to blank
• Repeat with a re-blank
• Drop 2 μL of the mixture on the nanodrop
• Measure on the nanodrop
• Save as well 2M Plate 1 sp20
• Hit measure

Bacterial Transformation of BBa_K542003

Purpose
Introduce a foreign plasmid into bacteria and to use those bacteria to amplify the plasmid in order to make large quantities of the plasmid. This is based on the natural function of a plasmid: to transfer genetic information vital to the survival of the bacteria.
The efficiency of the protocol is HIGHLY dependent on timing.
Conversions
100 ng of BBa_K542003 x 1 μL / 61.6 ng = 1.62 μL of gene
PROCEDURE

• Start thawing competent cells (E. coli top 10) on ice
• Competent cells are in the -80°C freezer
• Add 100 μL of competent cells into a 1.5 mL centrifuge tube
• Add 100 ng of DNA to the 1.5 mL centrifuge tube
• Incubate on ice for 30 minutes
• Heat shock cells in water bath for EXACTLY one minute
• Make sure the water level is high energy for the tube to float
• Adjust the temperature to 42°C
• Incubate on ice for five minutes
• Add 600 μL of psi broth to each centrifuge tube
• Psi broth is in the -4°C freezer
• Incubate centrifuge tubes in shaking incubator at 37°C at 200 rpm for two hours

Make three dilutions:

• Obtain two fresh centrifuge tubes and label them with the appropriate dilution
• 1:10
• 1:100
• 1:1000
• Add 90 μL of psi broth to each centrifuge tube BEFORE diluting
• Add 10 μL of transformed cells to the 1:10 dilution
• Mix the solutions by pipetting up and down multiple times
• Take 10 μL from the 1:10 solution and place it in the 1:100 dilution
• Repeat for the 1:1000 solution