Jan 05, 2022
Version 3
Cloning shRNA Oligos into pLKO.1 V.3
- 1Addgene
External link: http://www.addgene.org/tools/protocols/plko/
Protocol Citation: Addgene The Nonprofit Plasmid Repository 2022. Cloning shRNA Oligos into pLKO.1. protocols.io https://dx.doi.org/10.17504/protocols.io.b3hxqj7nVersion created by Addgene The Nonprofit Plasmid Repository
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 05, 2022
Last Modified: January 05, 2022
Protocol Integer ID: 56599
Abstract
This is the protocol accompanying the "pLKO.1 – TRC Cloning Vector". For information about the PLKO.1-TRC cloning vector and tips on designing shRNA oligos for pLKO.1 see Addgene's website: http://www.addgene.org/tools/protocols/plko/
Materials
STEP MATERIALS
NEBuffer 3 - 5.0 mlNew England BiolabsCatalog #B7003S
NEBuffer 1 - 5.0 mlNew England BiolabsCatalog #B7001S
AgeI - 300 unitsNew England BiolabsCatalog #R0552S
EcoRI - 10,000 unitsNew England BiolabsCatalog #R0101S
EcoRI - 10,000 unitsNew England BiolabsCatalog #R0101S
T4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
Protocol materials
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
NEBuffer 3 - 5.0 mlNew England BiolabsCatalog #B7003S
NEBuffer 1 - 5.0 mlNew England BiolabsCatalog #B7001S
AgeI - 300 unitsNew England BiolabsCatalog #R0552S
EcoRI - 10,000 unitsNew England BiolabsCatalog #R0101S
EcoRI - 10,000 unitsNew England BiolabsCatalog #R0101S
T4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S
NEBuffer 1 - 5.0 mlNew England BiolabsCatalog #B7001S
AgeI - 300 unitsNew England BiolabsCatalog #R0552S
EcoRI - 10,000 unitsNew England BiolabsCatalog #R0101S
EcoRI - 10,000 unitsNew England BiolabsCatalog #R0101S
T4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
Annealing Oligos
Annealing Oligos
Resuspend oligos in ddH2O to a concentration of 20 μM.
Add 5ul Forward oligo
5 µL
Add 5ul Reverse oligo
5 µL
Add 5ul 10X NEB Buffer 2
5 µL
Add 35 μL ddH2O
35 µL
Incubate for 4 minutes at 95°C in a PCR machine or in a beaker of boiling water.
00:04:00
Incubate the sample at 70°C for 10 minutes in a PCR machine.
00:10:00
Slowly cool to room temperature over the period of several hours.
03:00:00
Note
This will take a few hours, but it is important for the cooling to occur slowly for the oligos to anneal.
Note
If using a beaker of water, remove the beaker from the flame, and allow the water to cool to room temperature.
Digesting pLKO.1 TRC Cloning Vector
Digesting pLKO.1 TRC Cloning Vector
Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA)
6 µg
with 5 μL 10x NEB buffer 1
5 µL
NEBuffer 1 - 5.0 mlNew England BiolabsCatalog #B7001S
with 1 μL AgeI
1 µL
AgeI - 300 unitsNew England BiolabsCatalog #R0552S
Bring up to 50 μl with ddH2O
Incubate at 37°C for 2 hours.
02:00:00
Purify with Qiaquick gel extraction kit, eluting in 30 μL of ddH2O.
Digest eluate with EcoRI by mixing: 30 μL pLKO.1 TRC-cloning vector digested with AgeI
with 5 μL 10x NEB buffer for EcoRI
5 µL
EcoRI - 10,000 unitsNew England BiolabsCatalog #R0101S
with 1 μL EcoRI
1 µL
EcoRI - 10,000 unitsNew England BiolabsCatalog #R0101S
and 14 μL ddH2O
14 µL
Incubate at 37°C for 2 hours.
02:00:00
Run digested DNA on 0.8% low melting point agarose gel until you can distinctly see 2 bands, one 7kb and one 1.9kb.
Note
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.
Cut out the 7kb band and place in a sterile microcentrifuge tube.
Purify the DNA using a Qiaquick gel extraction kit. Elute in 30 μL of ddH2O.
Measure the DNA concentration.
Ligating and Transforming into Bacteria
Ligating and Transforming into Bacteria
Use your ligation method of choice. For a standard T4 ligation, mix: 2 μL annealed oligo from "Annealing Oligos" section above.
2 µL
With 20 ng digested pLKO.1 TRC-cloning vector from the "Digesting pLKO.1 TRC Cloning Vector" section above.
20 ng
Note
If you were unable to measure the DNA concentration, use 1 μL
With 2 μL 10x NEB T4 DNA ligase buffer
2 µL
T4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S
With 1 μL NEB T4 DNA ligase
1 µL
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
Bring up to 20ul with ddH2O
Incubate at 16°C for 4-20 hours.
04:00:00
Transform 2 μL of ligation mix into 25 μL competent cells, following manufacturer’s protocol.
Note
Due to the long terminal repeats found in lentiviral plasmids, we recommend using a strain that reduces the frequency of homologous recombination of unstable regions, such as Invitrogen Stbl3™ or NEB Stable cells. This will ensure that the repeats will be maintained and often results in a greater yield of DNA.
Plate on LB agar plates containing 100 μg/mL ampicillin or carbenicillin (an ampicillin analog).