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sgRNA plasmid cloning

Updated 1/19/2018 10:52 PM
Step-by-step
by Hannah Shen (created by Alexis Komor)

Introduction

Written by Alexis Komor from David Liu's lab.

Materials

  • T4 DNA ligase buffer (NEB)
    • T4 Polynucleotide Kinase (NEB)
      • Q5 Hot Start High-Fidelity 2X Master Mix (NEB)
        • DpnI (20U, NEB)
          • Buffer PB (Qiagen)
            • QIAprep spin column Qiagen)
              • DNA Wash Buffer (Omega Bio-tek)
                • Buffer EB (Qiagen)
                  • 2X QuickLigase Buffer (NEB)
                    • QuickLigase (NEB)

                      Procedure

                      Part 1: 5' phosphorylate both primers

                      1. Example set of primers to use for cloning. The 20 nt highlighted in green should be altered according to your protospacer sequence.

                      Universal reverse primer: GGTGTTTCGTCCTTTCCACAAG

                      EMX1 forward: GAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC

                      1. Combine the following components:
                      Table1
                      A
                      B
                      1
                      		ReagentVolume (uL)
                      2
                      		T4 DNA ligase buffer2
                      3
                      		Primer (100 uM stock solution)2
                      4
                      		H2O15
                      5
                      		T4 Polynucleotide Kinase1
                      6
                      		Total20
                      1. Incubate at 37°C for 20 m, heat inactivate at 95°C for 5 m.

                      Part 2: Perform PCR with phosphorylated primers

                      1. Combine the following components:
                      Table2
                      A
                      B
                      1
                      		ReagentVolume (uL)
                      2
                      		Q5 Hot Start High-Fidelity 2X Master Mix25
                      3
                      		Forward primer (10 μM stock solution, from Part 1)2.5
                      4
                      		Reverse primer (10 μM stock solution, from part 1)2.5
                      5
                      		pFYFsgRNA plasmid as template (50-200 ng/uL conc.)1
                      6
                      		H2O19
                      7
                      		Total50
                      1. PCR was performed according to the manufacturer’s instructions, using 67°C as an annealing temperature and a 50 s extension time.
                      1. Incubate the PCR product with DpnI at 37°C for 1 h.

                      Part 3: Purify PCR products

                      1. Combine 50µL PCR reaction mixture with 250µL Buffer PB
                      1. Load onto QIAprep spin column
                      1. Rinse with 750µL DNA Wash Buffer
                      1. Elute with 50µL Buffer EB

                      Part 4: Ligate the PCR products

                      1. Combine the following coponents
                      Table3
                      A
                      B
                      1
                      		ReagentVolume (uL)
                      2
                      		PCR product from Part 31
                      3
                      		H2O3
                      4
                      		2X QuickLigase Buffer5
                      5
                      		QuickLigase1
                      6
                      		Total10
                      1. Incubate at ambient temperature for 15 min.

                      Part 5: Transform the ligated product into competent cells of choice.

                      1. Transform 5 μL of the reaction solution into competent cells of choice.

                      Comments

                      Mohsin Badat
                      5/1/2018 04:31 PM
                      Hi, thanks for uploading this! Just wondering, where can I get the pFYFsgRNA plasmid from?

                      Best wishes
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