20.109(F16):Generate gRNA plasmid (Day3)

Introduction

CRISPR(i) details...

Protocols

Part 1: BE Communications Lab workshop

Part 2: Primer preparation

While you were away the sequences for the mutagenic primers you designed were submitted to Integrated DNA Technologies (IDT). IDT synthesized the primers then lyophilized (dried) them to a powder. Follow the steps below to resuspend your primers.

1. Centrifuge the tubes containing your lyophilized primers for 1 min.
2. Calculate the amount of water needed for each primer (forward and reverse, separately) to give a concentration of 100 μM.
3. Resuspend each primer stock in the appropriate volume of sterile water, vortex, and centrifuge.
4. Now prepare a dilution from your archival stock. Prepare 100 μL of a solution that has both the forward and reverse primers, each primer at 10 μM.
   • Try the calculation on your own first. If you get stuck, ask the teaching faculty for help.
   • Be sure to change tips between primers!
5. Return the rest of your primer stocks, plus your primer specification sheets, to the front bench.

Part 3: Complete sgRNA insertion and amplification reaction

We will be using the Q5 Site Directed Mutagenesis Kit from NEB to perform your site-directed mutagenesis reactions. Each group will set up one reaction, for your X#Z mutation. Meanwhile, the teaching faculty will set up a single positive control reaction, to ensure that all the reagents are working properly. You should work quickly but carefully, and keep your tube in a chilled container at all times. Please return shared reagents to the ice bucket(s) from which you took them as soon as you are done with each one.

1. Get a PCR tube and label the top with your mutation and lab section (write small!).
2. Add 10.25 μL of nuclease-free water.
3. Add 1.25 μL of your mutagenesis primer mix (each primer should be at a concentration of 10 μM).
4. Add 1 μL of IPC template DNA (concentration of 25 ng/μL).
5. Lastly, use a filter tip to add 12.5 μL of Q5 Hot Start High-Fidelity 2X Master Mix - containing buffer, dNTPs, and polymerase - to your tube.
6. Once all groups are ready, we will begin the thermocycler, under the following conditions:

Segment	Cycles	Temperature	Time
Initial denaturation	1	98 °C	30 s
Amplification	25	98 °C	10 s
		55 °C	30 s
		72 °C	2 min
Final extension	1	72 °C	2 min
Hold	1	4 °C	indefinite

• After the cycling is completed, the teaching faculty will complete the KLD reaction (which stands for "kinase, ligase, DnpI") using 1 μL of your amplification product, 5 μL 2X KLD Reaction Buffer, 1 μL KLD Enzyme Mix, and 3 μL nuclease-free water. The reactions will be incubated for 5 min at room temperature.

• The teaching faculty will then use 5 μL of the KLD reaction product to complete a transformation into an E. coli strain (NEB 5α cells of genotype fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17) that will amplify the plasmid such that you are able to confirm the appropriate mutation was incorporated. The transformation procedure will be as follows:

1. Add 5 μL of KLD mix to 50 μL of chemically-competent NEB 5α.
2. Incubate on ice for 30 min.
3. Heat shock at 42 °C for 30 s.
4. Incubate on ice for 5 min.
5. Add 950 μL SOC and gently shake at 37 °C for 1 h.
6. Spread 50 μL onto LB+Amp plate and incubate overnight at 37 °C.

Reagents

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