PCR & Insert target-sequence to pBX-083

PCR for target-sequence

Materials

• Taq DNA polymerase
• dNTPs
• Taq buffer 10*
• Forward primer（HIV-target、HBV-target-1、HBV-target-2）
• Reverse primer（None、HIV-target、HBV-target-2）
• Template DNA (pBX-083PB5-HS4-EF1A-EGFPnuc-2A-Bla-LoxN-BGpA-HS4-PB3)
• dd H2O

Methods

Point-mutation PCR for UAS

1. Centrifuge the primers 12000 rmp for 5 min.
2. Dissolve the primers into dd H2O to the concentration of 50p mol/μl. Subpackage the solution for about 50 μl per tube to avoid repeated freezing and thawing.
3. Add 1μl template DNA into 9μl dd H2O to dilute the template DNA.
4. The total volume is 250μl, including 125 μl Q5TM High-Fidelity 2X Master Mix (dilute it from 2X to 1X), 2.5μl forward primer, 2.5μl the first kind of reverse primer, 1 μl diluted template DNA and 119μl dd H2O. And divide the 250 μl solution into 5 PCR tubes in average. Attention, the operation should be finished on ice.
5. Set the PCR machine and run:

   Stage1: Initial denaturation: 98°C，30s.

   Stage2: Denaturation :98°C，10s

   Anneal: 57°C，60°C，63°C，respectively.15s.

   Extension: 72°C, 30s.

   30 cycles.

   Stage3: Final extension：72°C, 5 min.

   Hold: 4°C.

6. Check the product by gel eletrophoresis.
7. Keep the product in -20°C.

electrophoresis for the product of PCR

According to gel eletrophoresis, the PCR is successful under all anneal temperatures，and60°C have the best result.

Insert target-sequence to pBX-083

Materials

• NcoI、BspEI

Methods

1. Digestion for PCR product and pBX-083 skeleton as the usual protocol.
2. T4 ligase enzyme for PCR product and pBX-083 skeleton.
3. Transfection of product of ligase product.
4. Extract new plasmid

Results

Digestion for PCR product and pBX-083 skeleton as the usual protocol

PCR product digestion system Component Volume NcoI 0.5ul BspEI 0.5ul

DNA(23ng/ul) 8ul 10X NEBuffer 2.1 1ul Total 10ul

pBX-083 skeleton product digestion system Component Volume NcoI 0.5ul BspEI 0.5ul

DNA(450ng/ul) 1ul 10X NEBuffer 2.1 1ul H2O 7ul Total 10ul

Incuabte at 37C for 10 hours [9.22 23:00pm~9:00pm]

T4 ligase enzyme for PCR product and pBX-083 skeleton

We need five type of target-sequence as HIV1, HIV-HIV, HBV1, HBV2, HBV1-HBV2.

T4 ligase enzyme 1ul 10* T4 ligase enzyme Buffer 2ul Skeleton（7000bp）（45ng/ul） 4.5ul PCR product（1000bp）(18ng/ul) 4.5ul H2O 8ul Total 20ul

Transformation of the five ligase product to cell for pure plasmid

1.Immediately, place the tubes on ice, allow cell to thaw on ice, 10 min 2.Check the cell to see if they have thawed, gently flick the cells 1-2 times to evenly resuspend the cells. 3.Divide the competent cells into 5 tubes,50uL each tube, Mark them with HIV1, HIV-HIV, HBV1, HBV2, HBV1-HBV2. Keep the bacteria in ice during the procedure. 4.Add 20uL ligase mixture to each tube, shaking slightly 5.Incubate on ice for 30min 6.Heat shock, 42°C, 70s. 7.Keep on ice, 2 min 8.Add 200ul SOC medium to each tube, incubate 37°C, 200rpm, 45min 9.Centrifuge, 4000rmp, 5min, RT. Discard most of the medium and reserve 50uL. Resuspend. 10.Add 50uL bacteria to amp LB agar plate, distribute. 11.Incubate the Amp plates at 37°C, 16 hours.

Extract new plasmid

For the effiency of DH5α cell, only target-HIV ,target-HBV2,target-HBV1-HBV2 have colony. We transform the colony to 30 ml amp LB medium to OD=1.4,then we extract endo-free plasmid and no less than 500ng/ul.

Balance steps: 1. The column into the adsorption column CP4 (adsorption column in the collection tube) to join 500 mu l liquid equilibrium BL, 12000 RPM (~ 13400 x g) centrifugal 1 min, pour out collecting tube of liquid waste, will be put back into the collection tube adsorption column. 2. Take five to 15 ml overnight train bacteria liquid in centrifuge tube, 12000 RPM (~ 13400 x g) centrifugal 1 min, to absorb as much as possible In addition to the supernatant. 3. To allow bacteria to precipitate in the centrifuge tube to join 500 mu l solution P1 (please check whether has joined RNaseA), use Liquid move or vortex generator completely suspended bacterial cells to precipitate. 4. To join in centrifuge tube 500 mu l solution P2, gently upside down 6-8 times make bacteria sufficient cracking. 5. To join in centrifuge tube 500 mu l solution P4, gently flip 6-8 times up and down immediately, fully blending, appears white Floc, then placed about 10 min at room temperature, 12000 RPM (~ 13400 x g) centrifuge for 10 min, at this point in the At the bottom of the heart tube forming precipitation. 6. Will step on the collection of add in supernatant fluid filtration column CS (filter column in the collection tube), 12000 RPM (~ 13400 x g) 2 min, the centrifugal filtrate collection in clean 2 ml centrifuge tube (own). 7. To join in the filtrate of 0.3 times the filtrate volume isopropyl alcohol (isopropyl alcohol too much easy to cause pollution of the RNA), upside down After blending to adsorption column CP4 (adsorption column in the collection tube). 8. 12000 RPM at room temperature (~ 13400 x g) centrifugal 1 min, pour out collecting tube of liquid waste, put back the adsorption column to collect In the tube. 9. To add 500 mu l to protein adsorption column CP4 liquid PD, 12000 RPM (~ 13400 x g) centrifugal 1 min, pour out Header in the waste liquid, adsorption column CP4 to be included in the collection tube. 10. To join in the adsorption column CP4 drift lotion PW 600 mu l (please check whether has joined anhydrous ethanol), 12000 RPM 1 min (~ 13400 x g) centrifugal, pour out collecting tube of liquid waste, adsorption column CP4 to be included in the collection tube. 11. To join in the adsorption column CP4 600 mu l drift lotion PW, 12000 RPM (~ 13400 x g) centrifugal 1 min, pour out to collect Tube in the liquid. 12. The adsorption column CP4 put back into the collection tube, 12000 RPM (~ 13400 x g) centrifugal 2 min, aims to adsorption The column of floating lotion to remove residual. 13. The adsorption column CP4 put in a clean centrifuge tube, the middle parts of the adsorption film dangling add 100-300 mu l elution Liquid TB, place 2 min at room temperature, 12000 RPM (~ 13400 x g) centrifugal 1 min solutions of plasmid collected centrifuge in the tube

HIV:6000ng/ul HBV2:6600ng/ul HBV1-HBV2:6600ng/ul

All of these endo-free plasmid will be test in cell experiment