step1 Ordering Oligos Compatible whith pLKO.1

在前面的shRNA oligo设计中已经提到，在此不再赘述。

step2 Annealing oligos

1 resuspend oligos in ddH2O to concentration of 20uM，then mix：
因为我的oligo被稀释到了10uM，所以一下体系按照自己的浓度配比：

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.
（一般选择100度水浴锅5min，然后关掉自然冷却到室温，需要4-6个小时）

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）连接
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）转化
50ul感受态
全部链接产物
冰上30min
42度70s
马上放回冰上
放置5min
加1mlLB（无amp）37度摇床60min
3000rpm 4min离心，倒掉上清
取 50-100 μL 涂在含有氨苄青霉素（ 100 μg/mL ）的 LB 固体培养基上， 37℃ 倒置培养过夜。（如果涂上去的液体过多，则正置1h后倒置。

Step5 阳性克隆鉴定

PCR 验证，使用引物，见下篇文章“单克隆测序”
induciable plasmid kd 单克隆鉴定
mix：10
plko f：1
plko r：1
单克隆悬浮液：3
pcr 退火90s
跑胶：出现200kd左右条带为阳性

（2）测序