pLKOshRNA病毒质粒载体构建protocol方法详解(2)

5 μL 10x NEB buffer for EcoRI

1 μL EcoRI

14 μL ddH2O

> Incubate at 37oC for 2 hours.

4. Run digested DNA on 0.8% low melting point agarose gel until you can distinctly see 2 bands, one 7kb and one 1.9kb. Cut out the 7kb band and place in a sterile microcentrifuge tube.

When visualizing DNA fragments to be used for ligation, use

only long-wavelength UV light. Short wavelength UV light will

increase the chance of damaging the DNA.

5. Purify the DNA using a Qiaquick gel extraction kit. Elute in 30 μL of ddH2O.

6. Measure the DNA concentration.

C.4 Ligating and Transforming into Bacteria

1. Use your ligation method of choice. For a standard T4 ligation, mix:

2 μL

20 ng

2 μL

1 μL annealed oligo from step C.2. digested pLKO.1 TRC-cloning vector from step C.3. (If you were unable to measure the DNA concentration, use 1 μL) 10x NEB T4 DNA ligase buffer NEB T4 DNA ligase

干扰shRNA病毒质粒构建

to 20 μL ddH2O

> Incubate at 16oC for 4-20 hours.

2. Transform 2 μL of ligation mix into 25 μL competent DH5 alpha cells, following manufacturer's protocol. Plate on LB agar plates containing 100 μg/mL ampicillin or carbenicillin (an ampicillin analog).

D. Screening for Inserts

You may screen for plasmids that were successfully ligated by restriction enzyme digestion. However, once you have identified the positive clones, it is important to verify the insert by conducting a sequencing reaction.

D.1 Recommended Materials

D.2 Screening for Inserts

1. Innoculate 5 colonies from each ligation into LB + 100 μg/mL ampicillin or carbenicillin.

Day 2:

2. Spin down the cultures and use a miniprep kit to obtain DNA.

3. Conduct a restriction digest with EcoRI and NcoI:

1 μg

2 μL miniprep DNA 10x NEB buffer for EcoRI

0.8 μL EcoRI

0.8 μL NcoI

干扰shRNA病毒质粒构建

to 20 μL ddH2O

> Incubate at 37oC for 1-2 hours.

4. Run the digestion products on a 1% agarose gel. You should see two fragments, a 2kb fragment and a 5kb fragment.

5. Sequence positive clones with pLKO.1 sequencing primer (5' CAA GGC TGT TAG AGA GAT AAT TGG A 3').

You may need to adjust the sequencing conditions if the

DNA polymerase has difficulty reading through the

secondary structure of the hairpin sequence.

E. Producing Lentiviral Particles Before this step, you must contact your institution's Bio-Safety office to receive permission and institution-specific instructions. You must follow safety procedures and work in an environment (e.g. BL2+) suitable for handling HIV-derivative viruses.

For transient knockdown of protein expression, you may transfect plasmid DNA directly into the target cells. The shRNA will be expressed, but the DNA is unlikely to be integrated into the host genome.

For stable loss-of-function experiments, Addgene recommends that you generate lentiviral particles and infect the target cells. Addition of

puromycin will allow you to select for cells that stably express your shRNA of interest.

E.1 Recommended Materials

干扰shRNA病毒质粒构建

Note: pLKO.1 could also be packaged using pCMV-dR8.2 dvpr and pCMV-VSVG from the Robert Weinberg lab. For more information, visit Addgene's Mammalian RNAi Tools page.

E.2 Protocol for Producing Lentiviral Particles

This protocol is for transfection in a 6 cm plate. The protocol can be scaled to produce different amounts of virus as needed.

a. For each plasmid to be transfected, plate 7x105 HEK-293T cells in 5 mL of media in a 6 cm tissue culture plate. Incubate cells at 37oC, 5% CO2 overnight.

Although cells should regularly be passaged in DMEM +

10% FBS with penicillin/streptomycin, cells should be

plated at this step in DMEM + 10% FBS without antibiotics

(no penicillin or streptomycin).

b. Perform the transfection in the late afternoon because the

transfection mix should only be incubated with the cells for 12-15 hours.

c. In polypropylene microfuge tubes (do NOT use polystyrene

tubes), make a cocktail for each transfection:

1 μg pLKO.1 shRNA plasmid

750 ng psPAX2 packaging plasmid

250 ng pMD2.G envelope plasmid

to 20 μl serum-free OPTI-MEM

You may want to vary the ratio of shRNA plasmid,

packaging plasmid, and envelope plasmid to obtain the

ratio that gives you the optimal viral production.

干扰shRNA病毒质粒构建

d. Create a master mix of FuGENE® 6 transfection reagent in

serum-free OPTI-MEM. Calculate the amount of Fugene® and

OPTI-MEM necessary given that each reaction will require 6 μL FuGENE® + 74 μL OPTI-MEM. For example:

1x master mix: 6 μL FuGENE® + 74 μL OPTI-MEM

5x master mix: 30 μL FuGENE® + 370 μL OPTI-MEM

10x master mix: 60 μL FuGENE® + 740 μL OPTI-MEM

In a polypropylene tube, add OPTI-MEM first. Pipette FuGENE® directly into the OPTI-MEM - do not allow FuGENE® to come in contact with the walls of the tube before it has been diluted. Mix by swirling or gently flicking the tube. Incubate for 5 minutes at room temperature.

e. Add 80 μL of FuGENE® master mix to each tube from step c for a total volume of 100 μL. Pipette master mix directly into the liquid and not onto the walls of the tube. Mix by swirling or gently flicking the tube.

f. Incubate for 20-30 minutes at room temperature.

g. Retrieve HEK-293T cells from incubator. The cells should be 50-80% confluent and in DMEM that does not contain antibiotics. h. Without touching the sides of the dish, gently add

DNA:FuGENE® mix dropwise to cells. Swirl to disperse mixture evenly. Do not pipette or swirl too vigorously, as you do not …… 此处隐藏：5385字，全部文档内容请下载后查看。喜欢就下载吧 ……