Difference between revisions of "20.109(F16):Generate gRNA plasmid (Day3)"

Introduction

CRISPR(i) details...

Protocols

Part 1: BE Communications Lab workshop

Journal club...

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 insert the gRNA sequence into an expression vector. Each group will set up one reaction, for your insertion. 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 team color 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:

• 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.

Part 4: Journal article discussion

Reagents

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