VDL100.2 CLONING TRANSGENE INTO padenox

Transcription

1 1. Purpose 1.1. The purpose of this protocol is to transfer a transgene from the pshuttlex plasmid to padenox The starting material is 10 μg plasmid DNA This procedure is routinely performed in the Vector Development Laboratory (VDL) following Good Laboratory Practices (GLP). 2. Abbreviations and Definitions 2.1. SOP Standard Operating Procedure 2.2. VDL Vector Development Laboratory 2.3. GLP Good Laboratory Practices 2.4. BSA Bovine Serum Albumin 2.5. EDTA Ethylenediaminetetraacetic Acid 2.6. TE Tris-EDTA 2.7. LB Luria Broth 3. Equipment, Materials, and Reagents NOTE: All materials in contact with cells must be sterile, pyrogen-free and used according to the manufacturer s directions unless stated otherwise. Equivalent materials and equipment may be used but all changes must be recorded in the laboratory notebook Equipment Water bath set at 37 ºC Water bath set at 42 ºC Microcentrifuge Vortex Thermal Cycler Shaker Incubator 3.2. Materials ml thin-walled tubes Axygen Sterile pipet tips VWR ml tubes Axygen Cotton-plugged Pasteur pipets VWR Latex bulb for Pasteur pipets VWR ml snap cap tubes Falcon Page 1 of 7

2 cell spreader VWR LB/ampicillin plates Invitrogen 3.3. Reagents Deionized Water Baxter BD Adeno-X Viral DNA Clontech PI-SceI/I-CeuI Double Digest kit Clontech Restriction enzyme buffer SwaI New England Biolabs BSA New England Biolabs Phenol/Chloroform/Isoamyl Alcohol Invitrogen NaOAc Sigma M NaOAc VDL Absolute Ethanol % Ethanol VDL % Ethanol VDL mg/ml Glycogen Roche X TE buffer, ph 8.0 VDL DNA Ligase kit Invitrogen Stable 2 cells Invitrogen SOC Invitrogen LB Sigma 3.4. Starting Materials Plasmid DNA, 10 μg 4. Procedure PI-SceI/I-CeuI Digest 4.1. Prepare a 30 μl PI-SceI and I-CeuI double-digest of the recombinant pshuttlex plasmid DNA. Combine the reagents shown below in a 1.7 ml tube. PI-SceI/ I-CeuI Digest 10X Double digest buffer 3.0 μl Recombinant pshuttlex DNA 10 μg PI-SceI 2.0 μl I-CeuI 0.5 μl 10X BSA 3.0 μl Sterile water Q.S to 30 μl Page 2 of 7

3 4.2. Mix well and spin briefly to collect reagents at the bottom of the tube Incubate at 37 C for 3 hours. DNA Extraction 4.4. Add 70 μl 1X TE buffer to the digested plasmid DNA Add 100 μl phenol:chloroform:isoamyl alcohol to the digested plasmid DNA Vortex thoroughly Spin the tube in a microcentrifuge at 14,000 rpm for 5 minutes to separate the phases Carefully transfer the top aqueous layer to a clean 1.7 ml tube. Discard the interface and lower phase into an organic waste container Add 400 μl ice-cold 95% EtOH, 50 μl 3 M NaOAc, and 1 μl glycogen to the retained aqueous layer Vortex thoroughly Spin the tube in a microcentrifuge at 14,000 rpm for 5 minutes A white DNA pellet should be visible on the bottom of the tube Carefully remove and discard the supernatant Wash the pellet with ice-cold 70% ethanol Vortex thoroughly Spin in a microcentrifuge at 14,000 rpm for 5 minutes The DNA pellet should again be visible on the bottom of the tube Carefully aspirate off the supernatant Air dry the pellet for approximately 15 minutes at room temperature to evaporate residual ethanol When the pellet is dry, dissolve the DNA pellet in 10 μl sterile 1X TE buffer and store at - 20 C until use. Ligate transgene and padenox Prepare a 10 μl ligation reaction containing the transgene and padenox DNA. Combine the reagents in the table below in a thin wall 0.2 ml tube. Ligation Reaction Experiment Negative Control PI-SceI/I-CeuI digested plasmid DNA 2 μl --- BD Adeno-X Viral DNA 3 μl 3 μl 5X ligation buffer 2 μl 2 μl DNA ligase 1 μl 1 μl Page 3 of 7

4 Sterile water 2 μl 4 μl Gently mix then briefly spin in microcentrifuge to collect reagents at the bottom of the tube Transfer tubes to thermal cycler programmed to run overnight at 16 C. DNA Extraction Add 90 μl 1X TE buffer to the ligation reaction Add 100 μl phenol:chloroform:isoamyl alcohol to the ligation reaction Vortex thoroughly Spin the tube in a microcentrifuge at 14,000 rpm for 5 minutes to separate the phases Carefully transfer the top aqueous layer to a clean 1.7 ml tube. Discard the interface and lower phase into an organic waste container Add 400 μl ice-cold 95% EtOH, 50 μl 3 M NaOAc, and 1 μl glycogen to the retained aqueous layer Vortex thoroughly Spin the tube in a microcentrifuge at 14,000 rpm for 5 minutes A white DNA pellet should be visible on the bottom of the tube Carefully remove and discard the supernatant Wash the pellet with ice-cold 70% ethanol Vortex thoroughly Spin in a microcentrifuge at 14,000 rpm for 5 minutes The DNA pellet should again be visible on the bottom of the tube Carefully aspirate off the supernatant Air dry the pellet for approximately 15 minutes at room temperature to evaporate residual ethanol When the pellet is dry, dissolve the DNA pellet in 15 μl sterile deionized water and store at -20 C until use. SwaI digestion of non-recombinant padenox DNA Prepare a 20 μl digest for each experimental and control group as shown in the table below. SwaI Digest of Ligation Reaction Products Reagent Volume Ligation products (from 4.39) 15 μl 10X SwaI digestion buffer 2 μl Page 4 of 7

5 10X BSA SwaI Restriction Enzyme Total Volume 2 μl 1 μl 20 μl Incubate at 25 C for 2 hours. DNA Extraction Add 80 μl 1X TE buffer to the digestion reaction Add 100 μl phenol:chloroform:isoamyl alcohol to the ligation reaction Vortex thoroughly Spin the tube in a microcentrifuge at 14,000 rpm for 5 minutes to separate the phases Carefully transfer the top aqueous layer to a clean 1.7 ml tube. Discard the interface and lower phase into an organic waste container Add 400 μl ice-cold 95% EtOH, 50 μl 3 M NaOAc, and 1 μl glycogen to the retained aqueous layer Vortex thoroughly Spin the tube in a microcentrifuge at 14,000 rpm for 5 minutes A white DNA pellet should be visible on the bottom of the tube Carefully remove and discard the supernatant Wash the pellet with ice-cold 70% ethanol Vortex thoroughly Spin in a microcentrifuge at 14,000 rpm for 5 minutes The DNA pellet should again be visible on the bottom of the tube Carefully aspirate off the supernatant Air dry the pellet for approximately 15 minutes at room temperature to evaporate residual ethanol Resuspend pellet in 10 μl of sterile water Store at -20 C until use. Transform Stable 2 competent cells with recombinant DNA Thaw one vial of Stable 2 cells on ice Transfer 100 μl stable 2 cells to 14ml snap cap tube, pre-chilled on ice Add 1 μl recombinant DNA to cells and gently swirl Incubate cells and recombinant DNA on ice for 30 minutes Heat shock stable 2 cells for 25 second at 42 C Immediate transfer to ice and incubate for 2 minutes. Page 5 of 7

6 4.67. Add 0.9 ml room temperature S.O.C Place transformation reaction in a shaking 30 C incubator and shake at 225 rpm for 2 hours Prepare LB agar plates with ampicillin during this incubation Remove 4 LB agar + ampicillin plates from the 4ºC refrigerator and warm to 37ºC Transfer transformation reaction to a 1.7 ml tube Pellet cells by spinning in a microcentrifuge at 1000 x g for 2 minutes Remove all but 300 µl of the S.O.C Gently resuspend the pellet in the residual 300 µl of the S.O.C Apply 250 µl of the transformation reaction on one LB agar plate and 50 µl on two the second plate Spread cells across plate with sterile cell spreader Transfer plates to 30 ºC bacterial incubator for over night incubation Check plates for bacterial colonies Select colonies by numbering on the bottom of the plate with a marker Prepare one 14 ml snap cap tube for each colony selected Transfer 3 ml LB + ampicillin solution into each 14 ml snap cap tubes Number one 14 ml tube for each colony selected Inoculate media in each tube with the respective colony Return cap to tube but do not snap completely shut air needs to enter the culture for bacterial growth Place tubes in a 30 ºC shaking incubator for overnight incubation Prepare mini-prep DNA from cultures according to the SOP VDL706.1 Preparation of mini-prep DNA. 5. Data Collection and Management 5.1. A final report will be produced from the raw data and inserted into the laboratory notebook and a copy will be stored in the VDL permanent files Deviations of the protocol will be recorded in the laboratory notebook and on the Lab Meeting Sheet. 6. Review and Revisions Written by: Director, VDL Page 6 of 7

7 CENTER FOR CELL & GENE THERAPY Director, Vector Production Director, QA/QC Date Issued: 1/30/2006 Replaces VDL Annual Review: 2011 Reviewed without changes Changed and this version archived QA/QC by: Date: Issued: 7/30/11 Replaces VDL Reviewed without changes Changed and this version archived QA/QC by: Date: 2013 Reviewed without changes Changed and this version archived QA/QC by: Date: Page 7 of 7