20.109(F07): Phage by design, pt2

Introduction

Protocols

Part 1: M13.1 titers

The strains you transformed last time with M13K07 and M13.1 were inoculated into LB+Kan and grown overnight. If the two forms of the phage are equally robust, then the phage titer in the liquid surrounding the cells should be approximately the same. Ditto if the wild type strain, MG1655, and the MDS strain are equally capable of phage production. You will compare the PFU/ul of each culture to determine if this is true. The titering protocol is identical to the one from module 1. Since you don't know if the phage production will be improved or diminished by any of the variations, you will titer a wider-than-usual range of concentrations.

	phage = M13K07, strain = MG1655	phage = M13K07, strain = MDS	phage = M13.1, strain = MG1655	phage = M13.1, strain = MDS
10-6
10-8
10-10

You should also include a no phage control plate for confidence that the number of plaques you see next time arise as a result of your experiment.

Part 2: Phage deposition onto patterned slide

How to set up the ringstand for the deposition of the nanowires onto the ITO slide.

1. Clean the ITO slide by placing it into a 50 ml falcon tube with 1% Liquinox. Sonicate for 2 minutes. Remove the slide from the Liquinox solution with gloves or with tweezers and briefly rinse in Millipore H2O. Place the slide in a 50 mL Falcon tube with methanol. Sonicate for 2 minutes. Remove the slide and then let it air dry on a paper towel on the bench. Do not wipe the ITO surface.
2. Plasma clean the slide for 5 minutes. One of the teaching faculty will show you how to use the plasma cleaner.
3. While the slide is drying, set up the ringstand. Look at the figure to see how it should be set up. Hold the platinum electrode and the slide with metal tweezers which you can then fix using clamps.
4. Fill the beaker with 45 mL of Millipore H2O. Add 5 mL of the nanowire solution and mix with a pipette.
5. Lower the platinum mesh into the solution. Try to get as much of the platinum into the solution WITHOUT letting the tweezers touch the solution.
6. Lower the ITO slide into the solution with the ITO side facing the platinum. Make sure that your pattern is fully immersed in the solution (or else you won’t get deposition on your entire pattern), but do not let the tweezers touch the solution.

   

   Your platinum mesh and ITO slide should be parallel to each other.

7. The platinum electrode should be about ½ inch away from the ITO slide with the ITO side facing the platinum. Try to have the slide and the platinum parallel to each other.
8. Before you turn on the power supply, turn the voltage and the current all the way down (counter clockwise).
9. Attach the slide and the platinum mesh to the power supply using alligator clips. The negative should be hooked up to the platinum and the positive should be hooked up to the ITO. Also, the negative should be grounded on the power supply. See the figure.

   

   Make sure your connections match those in the graphic.

10. Turn on the power supply and slowly turn the voltage up to about 7.5 V. Keep an eye on the current. The current should not be above 0.01 amps. If the current is higher than that, turn down the current knob. If the current knob is all the way down, turn down the voltage. Use the multimeter to confirm that the charge is flowing by measuring the voltage difference between the two tweezers.
11. After 10 minutes, look at the solution. You should see a dark cloud collecting near the ITO slide. Turn off the voltage and slowly lift the ITO slide out of the solution. Use a 1 mL pipette to mix the solution.
12. Carefully return the ITO slide to the solution. Make sure that the tweezers are not touching the liquid. Turn the voltage back on. Check the voltage again using the multimeter.
13. After another 10 minutes, you should see a dark cloud collecting near the ITO slide. Turn off the voltage and slowly lift the ITO slide out of the solution. Look at the pattern. You should see a homogenous covering across the patterned part of the slide. If it is not fully covered, put the slide back in the solution for another 10 minutes. If it is covered, carefully set the slide, pattern side up, on a paper towel to dry for at least 1 hour.
14. Look at the slide and if there is any additional liquid on it, you can carefully wick away the liquid using the tip of a Kimwipe.

For next time

1. water
2. how to more densely pack nanowires