Agarose Gel electrophoresis

1. Preparing the agarose gel

Measure 1.0g of agarose powder and add 100 mL of TAE 1X;
Melt in a micro oven until solution becomes clear;
Let it cool;
Add 1µL Gelred;
Pour the melted agarose solution into the casting tray and let cool until it is solid.

2. Loading the gel

Add enough TAE 0.5 buffer so that there is about 2-3 mm of buffer over the gel;
Add 10X loading buffer to each PCR reaction;
Pipette 30µL each sample into separate wells in the gel;
Pipette 5µL of DNA ladder standard into at least one well.

3. Running the gel

Turn on the power supply to about 140 volts let it run for about 20 minutes.

4. Revelation

Put the gel under UV;
Make a copy of the image;
Get the gel containing the DNA we need.

PCR

1. Add the following component as listed below

Assemble all reaction components on ice and quickly transfer the reactions to thermocycle preheated to 94 °C and begin thermocycling.

2. Thermocycling conditions for a routine PCR

Preparation of LB Broth

1. add 10g Trytone;
2. add 5g yeast extract;
3. add 10g sodium chloride;
4. add 1L purified water;
5. use autoclave to sterilize.

Transformation

1. Put the tube of E. coli cells on ice until the last crystals disappear. And add 1-5 ul containing 1-100ng plasmid DNA to 50 ul of cells in a transformation tube on ice.
2. Place the mixture on ice for 30 minutes and never shake it.
3. heat shock at exactly 42℃ fors 90 seconds. Then pipette 500 ul LB into the mixture and put the tube at 37℃ for 60 minutes and shake vigorously.
4. warm the plates containing Ampicin to 37℃. Then spread the mixture onto the plate and incubate at 37℃ for 10-12 hours.
5. Picking up monoclonal colonies into 4ml LB broth containing Ampicin and put the tube at 37℃ for 8-10 hours and shake vigorously.
6. send the LB broth to bio-company to sequence the DNA plasmid. If the sequence is right, we can extract the plasmid from bacterial for next experiments.

Gel extraction

Gel extraction was performed according to the Universal DNA purification Kit(Cat. DP214-03).

Extraction and Purification of plasmid DNA

Plasmid extraction were carried out according to the GoldHi EndoFree Plasmid MAXI Kit. (Cat. CW2104M) for midiprep and TIANprep Mini Plasmid Kit.(Cat. DP103-03) for miniprep.

Site-Directed Mutagenesis

We used forward and reverse primers which prime at the same site and contain a mismatch at the specific base in terms of the original structure. Each primer contains 25-30 bases. And this mismatch defines the new sequence of the same site. Like PCR, the designing mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation. And we do all the procedure according to the Fast Mutagenesis System Kit(Code#FM111-01).

Ligation

1. Add all the components listed in the table above to a microcentrifuge tube.
2. Incubate the ligation mixture at 16℃ overnight.

Cell culture

All of the procedure about cells must be carried out under sterile conditions.

1. Resuscitation cell. Put the tube of frozen cells into 37℃ water within 1-2 minutes. Then transfer the cells into 15 ml falcon containing 10 ml of DMEM and centrifuge cells at 180g for 5 minutes at room temperature. Pour off the supernatant and use 8ml fresh DMEM (10% fetal bovine serum ) resuspend the cells. Transfer the cells to T75 flask and put the flask in a 37℃ incubator at 5% CO2.

2. Passage of the cells. Monitor the cell density daily. When culture reaches 80% confluence, cells should be passaged.
Prewarm the DMEM and trypsin-EDTA solution at room temperature. Remove the medium from the flask and wash cells once with 5ml of DMEM (with no FBS). Add 1 ml trypsin-EDTA into the flask. After 1-1.5 minutes, add 5ml of DMEM (with 10% FBS) and resuspend softly. Then transfer the cells into 15ml falcon and centrifuge at 150g for 5 minutes. Pour off the supernatant and use 14ml fresh DMEM (10% fetal bovine serum ) resuspend the cells. Transfer the cells to two T75 flasks and put the flasks in a 37℃ incubator at 5% CO2.

Establishing AAV-9

We used 293T cells to produce AAV-9. 293T cells express E1 gene products and they are commonly and extensively used for the production of E1-deleted AD viruses. All the procedure need to be done in biosafety cabinet.

Replace the DMEM in the flask with fresh DMEM medium before transfection. And add 10g carrier plasmid, 10g packaging plasmid, 20g helper plasmid and 1ml DMEM medium to a sterile centrifuge tube, and mix by pipetting. Add 120 μL of liposome transfection reagent and 1ml DMEM medium to another tube. After 5 minutes, mix the solution in the two tubes and incubate for 20 minutes at room temperature. Then add all the solution into flask, gently shake the dish to mix the liquid. And incubate the cells in a 37℃ incubator at 5% CO2 for 12 hours. Then replace the DMEM in the flask with 12ml fresh DMEM (with 10% FBS) and incubate for 48 hours.

Collect the supernatant and cells in the flask into different centrifuge tube. Take freeze-thaw cycle in a liquid nitrogen and 37 ° C water for 3 times., centrifuge at 10000 rpm for 10 minutes., and collect the supernatant. Mix the supernatant and purify with a 0.45μm filter. Add 1M NaCl and 10% PEG 8000 solution in half volume of the supernatant to the supernatant, mix well and put it in 4℃ overnight. After centrifugation at 12,000 rpm for 2 hours, the supernatant was discarded, and the precipitated virus was dissolved in a PBS solution, and sterilized by filtration using a 0.22 μm filter. Add 0.1μL enzonase versa nuclease to each 1 mL of crude virus extract to remove residual plasmid DNA, Incubate the mixture at 37 ° C for 1 h. Take the supernatant after centrifuging at 1000 rpm for 10 min. Purify the extract with Biomiga Adeno-associated virus purification kit.