20.109(S14):Complete Western and prepare damaged DNA (Day4)

Introduction

More coming Wednesday. Thanks for your patience!

Brief reminder of where we are. Then…

Topic 1: gel purification and perhaps idea of diagnostic digestion more broadly

Topic 2: western secondary considerations and visualization options

Protocols

Today you get to experience grad student life, juggling multiple assays blah blah blah...

Part 1: Digest plasmid for NHEJ assay

• You will digest pMax-BFP-MCS at the cut site(s) that you chose to investigate last time. We will then evaluate and purify the DNA using gel electrophoresis.
• To avoid pipetting very small volumes, you will either prepare a reaction cocktail that uses no less than 1 μL of any restriction enzyme, or you will prepare an intermediate dilution of said enzyme(s).
• Note that enzyme stock concentrations can be found on the NEB product page for that enzyme.

1. By whichever approach outlined above, combine 3.5 μg of DNA with water, buffer, and enzyme in a well-labeled eppendorf tube. Whether you prepare an enzyme dilution or a master mix, the enzyme should be added last.
   • Why? What would happen if you added the enzyme directly to water?
   • Recall that you are using 2.5 U of each enzyme per μg of DNA.
2. Flick the tubes to mix the contents, touch-spin, then incubate the mixtures at 37°C for at least one hour. Write down your start time and also set a timer, in case you get distracted later on.
   • While your samples are digesting, you can finish the Western. (Or at least start finishing it!)

Part 2: Complete Western protein assay

A week ago, you prepared protein extracts from K1 and xrs6 gels, separated them by SDS-PAGE, and transferred them to a nitrocellulose membrane. On that day, blots were moved to blocking buffer. Four days later, they were moved to plain PBS. Finally, late yesterday, they were incubated with primary α-Ku80 antibody overnight at 4 °C. (See reagent list at end of lab for concentrations/compositions/etc.)

1. Obtain your blots from the front bench. Pour the antibody solution into a conical tube, writing the identity of the antibody and the date on the tube.
   • Because the antibody is in excess, sometimes the primary solution may be re-used on another blot. Worth saving until we see how our Westerns come out today, at least!
2. Add enough TBS-T to cover your membranes – between 10-15 mL should work, but you don't need to measure out this volume. Keep in mind that the washing steps work by dilution, so it is a balance between adding enough to create a sink for the primary antibody, but not so much that you make a huge mess on the shaker!
   • TBS-T stands for Tris-buffered saline with Tween 20 (a surfactant).
3. Shake your container for 5 min at 80 rpm, using the room temperature shaker in fume hood.
4. Repeat for a total of 3 washes.
5. Just before pouring off the last wash, prepare the secondary antibody. Specifically, prepare a 1:3000 dilution of GAR-AP in TBS-T at a total volume of 9 mL.
   • GAR-AP stands for goat anti-rabbit--alkaline phosphatase conjugate.
6. Shake at 80 rpm for about 45 minutes.
7. Wash the blot as before, for 3 washes.
8. Once you start the third wash, begin to prepare your development solution. Add 1 mL of development buffer stock to 24 mL H2O. Next, add 250 μL of reagent A and 250 μL of reagent B to the well-mixed buffer.
9. After removing the last TBS-T wash, add about half the development solution to your blot; save the rest for now.
10. Put your blot on the shaker. Check it about every 10 minutes. Full development should take somewhere between 20 and 60 minutes, perhaps a little longer.
    • Be sure you know what size band you are looking for! There are likely to be some cross-reactive/off-target/non-specific bands as well.
11. Once you are satisfied with the look of your bands, remove the development solution and add distilled water. Shake the blot for a final 10 minutes.
12. Finally, move the blot on top of a piece of filter paper to dry. Either you or the teaching faculty will take and post pictures of these on the wiki, depending on whether they appear ready by the end of lab.

Part 3: Gel purify digested plasmid

For next time

revising microbiota summaries, mainly

cell doubling calculation for fun

first steps of lipofection calculation? (suggestion though, not collected?)

Reagent list

write something here or not accessible to edit

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