step1 Ordering Oligos Compatible whith pLKO.1

在前面的shRNA oligo設(shè)計中已經(jīng)提到，在此不再贅述。

step2 Annealing oligos

1 resuspend oligos in ddH2O to concentration of 20uM，then mix：
因為我的oligo被稀釋到了10uM，所以一下體系按照自己的濃度配比：

10ul Forward oligo

10ul Reverse oligo

5ul 10XNEB buffer2

25ul ddH2O

2 two ways you can choose
（1）if you used PCR machine,
4min 95℃
10min 70℃
then slowly cool to room temperature over the period of several hours.
(2) if using a braker of water
4min in boiling water
then remove the breaker from the flame,and allow the water to cool to room temperature. This will take a few hours, but it is important for the cooling to occur slowly.
（一般選擇100度水浴鍋5min，然后關(guān)掉自然冷卻到室溫，需要4-6個小時）

Step3 Digesting PLKO.1 Cloning Vector

(1)Digest with Agel.

6ul PLKO.1 vector

5ul 10x NEB buffer 1

1ul Agel

to 50ul ddH2O

incubate at 37℃ for 2h

（2）purity with Qiaquick gel extraction kit. Elute in 30 ul of
ddH2O

(3)digest with Ecol:

30ul pLKO digest with Agel
5ul 10xNEB buffer for Ecol
1ul EcoR
to 14ul hhH2O
incubate at 37℃ for 2h

（4）Run digested DNA on 0.8% low melting point agarose gel. you can see 2 bands,one 7kb and one 1.9kb. Recycle 7kb band.

(5) purify the DNA using a Qiaquick gel extraction kit. Elute in 30ul of ddH2O.

(6) Measure the DNA concentration.

step 4 ligating and transforming into bacteria

（1）連接
4ul oligo from step2
20ng (or 1ul) PLKO step3
2ul 10x NEB T4 DNA ligase buffer
1ul NEB T4 DNA ligase
to 20ul ddH2O
incubate at 16℃ for 4-20h
（2）轉(zhuǎn)化
50ul感受態(tài)
全部鏈接產(chǎn)物
冰上30min
42度70s
馬上放回冰上
放置5min
加1mlLB（無amp）37度搖床60min
3000rpm 4min離心，倒掉上清
取 50-100 μL 涂在含有氨芐青霉素（ 100 μg/mL ）的 LB 固體培養(yǎng)基上， 37℃ 倒置培養(yǎng)過夜。（如果涂上去的液體過多，則正置1h后倒置。

Step5 陽性克隆鑒定

PCR 驗證，使用引物，見下篇文章“單克隆測序”
induciable plasmid kd 單克隆鑒定
mix：10
plko f：1
plko r：1
單克隆懸浮液：3
pcr 退火90s
跑膠：出現(xiàn)200kd左右條帶為陽性

（2）測序