20.109(F16):Generate gRNA plasmid (Day3)

Introduction

CRISPR(i) details...

Protocols

Part 1: BE Communication Lab workshop

Our communication instructors, Dr. Sean Clarke and Dr. Diana Chien, will join us today for a workshop on preparing and delivering your Journal Club presentation.

Part 2: gRNA oligo preparation

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

1. Centrifuge the tube containing your lyophilized gRNA oligo for 1 min.
2. Calculate the amount of water needed to give a stock concentration of 100 μM.
3. Resuspend each primer stock in the appropriate volume of sterile water, vortex, and centrifuge.
4. Calculate the volume of your stock that is required to prepare a 100 μL of solution that contains your gRNA oligo at a concentration of 10 μM.
   • Try the calculation on your own first. If you get stuck, ask the teaching faculty for help.
5. Recall from the M2D1 exercise that an amplification reaction requires two primers. The gRNA oligo will serve as the forward primer in the insertion and amplification reaction and a universal CRISPRi oligo will be the reverse primer.
   • Obtain an aliquot of the CRISPRi reverse primer (resuspended at a concentration of 100 μM) from the front bench.
6. Prepare a primer mix that contains both your gRNA oligo (or 'primer') and the CRISPRi reverse primer at a final concentration of 10 μM in 100 μL of sterile water.
   • Be sure to change tips between primers!
7. Return the rest of your gRNA oligo stock, plus your primer specification sheet, to the front bench.

Part 3: Complete gRNA 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 primer mix (each primer should be at a concentration of 10 μM).
4. Add 1 μL of pgRNA plasmid 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 insertion (or '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

We will start today with a discussion of a research article by Otoupal et al. entitled "CRISPR perturbation of gene expression alters bacterial fitness under stress reveals underlying epistatic constraints". In their research, the authors completed

...

Our paper discussion will be guided by all that you have learned about how to write a cohesive story that clearly reports the data and provides strong support for the conclusions made about the data. During the paper discussion, everyone is expected to participate - either by volunteering or by being called upon!

Introduction

Remember the key components of an introduction:

• What is the big picture?
• Is the importance of this research clear?
• Are you provided with the information you need to understand the research?
• Do the authors include a preview of the key results?

Results

Carefully examine the figures. First, read the captions and use the information to 'interpret' the data presented within the image. Second, read the text within the results section that describes the figure.

• Do you agree with the conclusion(s) reached by the authors?
• What controls are included and are they appropriate for the experiment performed?
• Are you convinced that the data are accurate and/or representative?

Discussion

Consider the following components of a discussion:

• Are the results summarized?
• Did the authors 'tie' the data together into a cohesive and well-interpreted story?
• Do the authors overreach when interpreting the data?
• Are the data linked back to the big picture from the introduction?

Reagents

Navigation links

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