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. Aliquotted volumes are presented for a long term storage to reduce the loss of viral titer through multiple freeze-thaw cycles. 1
17. A second harvest can be carried out by adding 10 mL of complete medium to the cells after the first harvest and incubating at 37°C for a further 24 hours. The first harvest can be stored at 4°C overnight to allow the second harvest to be added to it the following day (freezing the supernatant would result in a greater loss of titer).
18. Collect the second supernatant on Day 5 (as in steps 11-13) and combine this with the first harvest.
19. For viral titers that are 10⁶ IU/mL and higher, you can quickly and easily titer your virus preparation using the qPCR Lentivirus Titer Kit (LV900) available from abm.
    In addition, our Ultra-Pure, Lentiviral Purfication Kit (LV998) will allow you to concentrate the virus to a higher titer if desired.

Procedure:

1. Plate the target cells in a 24-well plate, 24 hours prior to viral infection at a density of 0.5×10⁵ cells per well. Add 0.5 ml of complete optimal medium (with serum and antibiotics if required) and incubate the cells at 37°C with 5% CO2 overnight.
2. Prepare a mixture of complete media with polybrene at a concentration of 8 μg/ml. Remove the growth media from the wells and replace with 0.5 ml of the polybrene-media-mix per well (adjust volume as necessary if using a different size plate). If the transduction efficiency of the target cells is low, add in ViralPlus Transduction Enhancer G698 at 1:100 (or your own optimized dilution ratio).
3. Once an effective MOI has been determined for the target cells through preliminary test infections, use the appropriate volume of virus to infect your cells. You should include a transduction well with a positive GFP control virus and an appropriate blank control viral construct. Leave one well of uninfected cells as an additional standard control. Following the infection, incubate the cells at 37°C with 5% CO2 overnight
4. Remove the culture medium and replace with 1 ml of complete medium. Incubate the cells at 37°C with 5% CO2 overnight.
5. The following day, split the cells 1:3 or 1:5 (depending on the growth rate of your target cells) and continue incubating for 48 hours in complete media.
6. The infected cells can then be selected for stable expression using appropriate antibiotic selection at a minimum concentration, as determined by a killing curve. Downstream expression can then be assayed by a number of techniques, including Western blot or RT-PCR.

Procedure:

1. Resuspend the target cells in fresh pre-warmed complete culture medium at concentration of 10⁵ -107 cells/ml in a final volume of 8 ml. Aliquot 2 ml into each of 4 x15 ml sterile conical tubes.
2. Once effective MOI has been determined for target cells through preliminary test infections, use the appropriate volume of virus to infect cells with polybrene at concentration of 8 μg/ml. If transduction efficiency of target cells is low, add in ViralPlus Transduction Enhancer G698 at 1:100 (or at own optimized dilution ratio). You should include a transduction well with a positive GFP control virus and an appropriate blank control viral construct. Leave one conical tube as uninfected cells as an additional standard control.
3. Gently mix and incubate cells for 20 minutes at room temperature in the tissue culture hood.
4. Centrifuge cells for 30 minutes at 800 x g at 32°C.
5. Remove virus containing medium and resuspend cell pellet with 2 ml of fresh complete culture media. In a 6-well plate, transfer each suspended cell pellet into its own well. Incubate cells for 18-72 hours.
6. Transfer cells into separate sterile 15 ml conical tubes, and centrifuge for 5 minutes at 200 x g. Aspirate media and replace with 2 ml complete media (with appropriate selection antibioticoptional). Transfer cells into separate tissue culture plates, and incubate overnight.
7. The following day, split cells 1:3 or 1:5 (depending on the growth rate of your target cells) and continue incubating for 48 hours in complete media.
8. The infected cells can then be selected for stable expression using appropriate antibiotic selection at a minimum concentration, as determined by a killing curve. Downstream expression can then be assayed by a number of techniques, including Western blot or RT-PCR.