Difference between revisions of "Team:TP-CC San Diego/Protocols"

Procedure:

1. Find gene of interest
2. Used exon 1 and exon 8 of EGFR gene because exon 2-7 has mutations
3. Input into crispr gRNA design tool: http://crispr.mit.edu
4. Review possible off target sights and mismatches
5. Choose guides that have less off target sights and all sights have at least 3 mismatches

Procedure:

1. Place reaction in a thermocycler under following conditions:
2. 37˚C 30 mins
3. 95˚C 5 mins and then ramp down to 25˚C at 5˚C/min
4. Before Ligation, dilute annealed oligos at 1:200 in EB

Materials:

1. 1uL cut plasmid
2. 1uL oligo
3. 1uL 10X T4 ligation buffer
4. 6uL H2O
5. 1uL Ligase

Procedure:

1. Combine all materials in a test tube.
2. Place at room temperature for one hour.

Procedure:

1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting.
7. Centrifuge for 30–60 s and discard the flow-through.
8. Wash the QIAprep 2.0 spin column by adding 0.75ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through. Transfer the QIAprep 2.0 spin column to the collection tube. Centrifuge for 1 min to remove residual wash buffer.
9. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

Procedure:

1. In the afternoon, seed ~1.2 x 107 293T cells in a 10 cm dish.
2. Check to make sure the cells are 70-80% confluent.
3. For each 10 cm dish prepare the transfection as follows:
   Solution A: Dilute 20 μg DNA plasmids (10 μg expression vector and 10 μg of abm's Second Generation (LV003) or Third Generation (LV053) Packaging Mix) in 1 mL serum-free, antibiotic- free medium.
   Solution B: Dilute 20 μg of LentiFectin Transfection reagent (G074) in 1 mL sereum-free, antibiotic-free medium.
4. Incubate both solutions at room temperature for 5 minutes.
5. Mix Solutions A and B together well and incubate at room temperature for 20 minutes. This will create the transfection complex.
6. Add 4.5 mL serum-free medium to the transfection to complex.
7. Remove medium from the cells in the 10 cm dish.
8. Add the complete transfection complex to step 4 to the cells and incubate at 37˚C for 5-8 hours. Avoid dislodging the cells by gently adding the mixture against the side wall of the dish.
9. Add 0.65 mL FBS to the 10 cm dish and incubate at 37˚C overnight.
10. Remove the transfection medium from the cells.
11. Add 10 mL complete culture medium to the cells.
12. Incubate at 37˚C for 24 hours.
13. Collect the supernatant medium from the culture dish.
14. Centrifuge the supernatant at 3000 rpm at 15 minutes at 4˚C to the pellet cell debris.
15. Transfer the cleared supernatant to a fresh tube. Filter the cleared supernatant with a low-protein binding 0.45 μM sterile filter.
16. The viral titer of the first harvest is approximately 10⁶ IU/mL. The filtered supernatant will be ready for In vitro infections or further concentration and/or purification. Alternatively, it can be stored at -80˚C as viral stock for future applications.