試劑
實(shí)驗(yàn)步驟
1.合成Nestin干擾序列
2.干擾序列退火
將正反向干擾序列以10μM濃度溶于ddH2O,，按以下體系將正反向干擾序列退火形成雙鏈粘末端DNA：
10 μL Forward oligo
10 μL Reverse oligo
5 μL 10x NEB buffer 2
25 μL ddH2O
置于100度水，自然降溫
3.將Nestin干擾序列克隆至PLKO.1載體質(zhì)粒
3.1 PLKO.1載體質(zhì)粒酶切
AgeI HF+ EcoRI HF酶切pLKO.1載體質(zhì)粒,，體系如下：
37°C孵育1.5h,。(酶切時(shí)間可延長(zhǎng)到＞2h)
瓊脂糖凝膠電泳，可見1kb和7kb兩個(gè)條帶,，切膠回收7kb條帶（靠凹槽的那條）,。
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.
1,、配制成1%瓊脂糖膠（0.5小時(shí)）0.7g+70ml 1TAE溶液（濃度越大,，區(qū)分度越小）,，加10000的核酸染劑（amino acid stain）7μl,。
2、電泳槽中,，黑色為負(fù)電極,，紅色為正電極，凹槽對(duì)著負(fù)極,。大凹槽可加50μl,，小凹槽可加10μl。
3,、吸取5μl DL5000 DNA marker加入電泳槽,。
4、吸取5μl 10*DNA loading buffer加入PLKO酶切液中,。
5,、參數(shù)設(shè)定100V，20min左右（條帶跑到膠中間比較合適）,。
6,、暴光200ms以上，觀看條帶,。
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.
7,、剪下條帶，稱重,。專用試劑盒,，用1：1溶解液溶解（即若條帶重220ug，則用220ul溶解液）,。60ul洗滌液洗2次,，離心，去除下清液,，甩干,，加25ul DDH2O溶解,，收集下清液。
8,、測(cè)量雙酶切載體質(zhì)粒濃度
3.2 連接
體系如下：
(If you were unable to measure the DNA concentration, use 1 μL)
雙酶切載體質(zhì)粒要測(cè)濃度,，再算體積。
16°C孵育4-20h,。
3.3 轉(zhuǎn)化
從-80℃冰箱取出DH5α感受態(tài)菌100ul（一般用20ul即可）,，立即放冰上緩慢溶解10min。
把2μL連接產(chǎn)物加入到感受態(tài)細(xì)胞中,，放置冰上孵育30min,。
于42℃熱激細(xì)胞30s~60s。
熱激完成后,，立即將細(xì)胞轉(zhuǎn)移到冰上孵育2min,。
加入250μL S.O.C. medium，然后在37℃,、225 RPM的搖床里孵育1h,。離心，即其中100ul,，吹打細(xì)胞溶解,。
把100μL轉(zhuǎn)化物涂板至100（50) μg/mL Amp的LB平板上，37℃孵育過(guò)夜,。（倒置培養(yǎng),，即字在上面）
挑選出單菌落，選出重組子進(jìn)行雙酶切檢測(cè)和測(cè)序,。(挑出單菌落到LB + 100 μg/mL ampicillin的15ml離心管中,，取1ml送測(cè)序)
4.慢病毒包裝與轉(zhuǎn)染
4.1慢病毒包裝方法：
a. 將293T細(xì)胞接種在10cm皿中，待其90%滿的時(shí)候換新鮮不含雙抗的293T培養(yǎng)液（DMEM+10%FBS),。
b.混合慢病毒載體質(zhì)粒,目的質(zhì)粒
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
c.(最好在下午或者晚上做)步驟a 2h后將慢病毒載體質(zhì)粒,，目的質(zhì)粒混合加入無(wú)血清的OPTI-MEM培養(yǎng)基中（先加培養(yǎng)基,，再加Lipo-fectamine 3000,，最后加質(zhì)粒。Lipo-fectamine 3000與質(zhì)粒1：1加入,，如2ul: 2ug,。質(zhì)粒需經(jīng)驗(yàn)性計(jì)算用量）Lipo-fectamine3000說(shuō)明書推薦2種用量，可參考,。
（靜置5min后加入X-treme HP,，非常規(guī)），顛倒混勻后靜置15min,，加入293T細(xì)胞中,。
d. 12-16h左右換液( fresh DMEM + 10% FBS + penicillin/streptomycin),，繼續(xù)培養(yǎng)24h-48h左右，收集含有病毒的上清培養(yǎng)液(polypropylene storage tube),Spin media at 1,250 rpm for 5 minutes to pellet any HEK-293T cells that were inadvertently collected during harvesting.儲(chǔ)存于4℃冰箱
e. 慢病毒超速離心濃縮,，棄上清,，溶于1×PBS后，分裝保存于-80℃,。（可以不離心,，直接用上清培養(yǎng)液加入，一般是1ml培養(yǎng)液加1ml培養(yǎng)基）
4.2Determining the Optimal Puromycin Concentration：
Each cell line responds differently to puromycin selection. Addgene strongly recommends that you determine the optimal puromycin concentration for your cell line before initiating your experiment.
Day 1: a. Plate target cells in ten 6 cm plates and grow at 37° C, 5% CO2 overnight.
Day 2: b. The target cells should be approximately 80-90% confluent.
c. Dilute puromycin in the preferred culture media for your target cells. The final concentration of puromycin should be from 1-10 μg/mL in 1 μg/mL increments. d. Label plates from 1-10 and add appropriate puromycin-containing media to cells.
Days 3+: e. Examine cells each day and change to fresh puromycin-containing media every other day.
f. The minimum concentration of puromycin that results in complete cell death after 3-5 days is the concentration that should be used for selection in your experiments. (You may wish to repeat this titration with finer increments of puromycin to determine a more precise optimal puromycin concentration.)
4.3慢病毒轉(zhuǎn)染方法：
準(zhǔn)備六孔板一個(gè)孔細(xì)胞,，吸去細(xì)胞培養(yǎng)液,，加入混勻了的病毒轉(zhuǎn)染液（950μL培養(yǎng)基+50μL濃縮病毒+1μL 1000×Polybrene），37℃,，5% CO2培養(yǎng)箱中培養(yǎng),。12h后，更換回普通培養(yǎng)基,。使用Puro（PLKO.1載體）或zeocin（pENTR223.1載體）篩選3~5天，純化轉(zhuǎn)染成功細(xì)胞株,。PLL3.7載體轉(zhuǎn)染后,，通過(guò)流式細(xì)胞儀分選GFP+細(xì)胞，純化轉(zhuǎn)染細(xì)胞株,。
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