Winnie Chang

Construct pLKO.1 shRNA plasmids for gene knockdown
1. Vector backbone(1) pLKO.1-TRC cloning vector -Puro (Addgene, #10878)(2) pLKO.1-tRFP657 (derived from (1) but exchange Puro into tRFP657)
2. Design shRNA oligos for pLKO.1
Determine 21-mer target in your gene using Predesigned shRNA (MERCK)
https://www.sigmaaldrich.com/JP/en/semi-configurators/shrna?activeLink=productSearch-> Search Genes/Clones: key in your interest gene symbol -> search-> Select shRNA Clones: choose the species and click details. Ex:

Choose validated knockdown efficiency > 90%
Copy Target Sequence as 21-mer as sense, and use complementary sequences as antisense
3. Order Oligos Compatible with pLKO.1
To generate oligos for cloning into pLKO.1, insert your sense and antisense sequences fromstep 2 into the oligos below. Do not change the ends; these bases are important for cloning
the oligos into the pLKO.1 TRC-cloning vector.
Forward oligo:
5’ CCGGCCC–21bp sense–CTCGAG–21bp antisense–TTTTTGGAAAT 3’
Reverse oligo:
5’ AATTATTTCCAAAAA–21bp sense–CTCGAG–21bp antisense–GGG 3’
Order oligos with 100 M DDW

Ex: for human SETDB1
Forward oligo:
CCGGCCCGCTCAGATGATAACTTCTGTACTCGAGTACAGAAGTTATCATCTGAGCTTTTTGGAAAT
Reverse oligo:
AATTATTTCCAAAAAGCTCAGATGATAACTTCTGTACTCGAGTACAGAAGTTATCATCTGAGCGGG

Winnie Chang

4. Cloning Oligos into pLKO.1
The pLKO.1-TRC cloning vector contains a 1.9 kb stuffer that is released upon digestion withEcoRI and AgeI(1) Digesting pLKO.1 vectorVector (6 g)
10x rCutSmart

6 l

EcoRI

1 l

AgeI

1 lDDW46 l

Total

60 l

6 l

Incubate at 37oC for 2 hours and run agarose gel(2) Annealing oligos
Forward oligo (100 M)

1 l

Reverse oligo (100 M)
10x NEB buffer 21 l

DDW

43l

Total

50 l

5 l

95oC 4 min
70oC 10 min
Take out the heat block and cool it down to room temperature (at least 1 hour)(3) Run 0.8% agarose gel (100V, 30 min) with step 4-1 digested vectors
Expected fragment size: 1.9 kb (stuffer), 7 kb (cut vector)-> Cut 7 kb digested vector and do gel extraction-> Elute DNA with 25 l DDW(4) Ligation*10x dilution for annealed oligos: 5 l mixture (from step 4-2) + 45 l DDW
10x diluted oligos
3 lPurified vector (step 4-3)

1 l

2x mighty ligation mix

4 lTotal

8 lIncubate at 16oC for 1-3 hours

Winnie Chang(5) Transformation (DH5)-> Take 3 l of ligation product (step 4-4) into 25 l of DH5-> on ice 30 min -> heat shock 42oC for 45 sec -> on ice 3 min -> add 80 l SOC-> all for plating with Amp plate(6) Mini-culture
Pick up 4 colonies each group for mini-culture (2 ml LB+2 l Amp)
(7) Mini-prep
Dissolve DNA with 30 l TE(8) Enzyme digestion check
Mini-prep DNA

2 l

10x buffer M

2 l

PvuII

0.3 l

DDW

15.7 l

Total

20 l

(9) Run 1% agarose gel (100V, 20 min)Correct expected fragment size: 3712, 2513, 776 bp
Negative control of original vector cutting size: 2810, 2604, 2513, 776, 198 bp(10) Select correct plasmids for sequencing*Sequencing method: dGTP
Select Premix1 sequencing and note dGTP 使用 to 特記事項欄【Premix1 シングル解析】¥2,000 / サンプル
テンプレート DNA と Primer を混合したサンプルをご提供いただきます。
シーケンサーにて 2 時間の泳動を行い、ご提供の base 数は 1000bp 程度になります。
Web サイトより Premix1 シングル解析をご選択いただき、「特記事項欄」に、「dGTP 使用」と予めご記入いただきますと、シーケンス反応時に弊社にて dGTP試薬を使用して解析を実施します。