VDL101.3 CLONING TRANSGENE INTO pad5f35

Transcription

1 Purpose 1.1. The purpose of this protocol is to transfer a transgene from the pshuttlex plasmid to pad5/f 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 2.8. TAE Tris Acetate, 0.05 EDTA 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 Spectrophotometer 3.2. Materials ml thin-walled tubes Axygen Sterile pipet tips VWR ml tubes Axygen ml snap cap tubes Falcon Page 1 of 9

2 Cell spreader VWR LB/ampicillin plates Invitrogen 3.3. Reagents Deionized Water Baxter 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 Stbl2 competent cells Invitrogen SOC Invitrogen LB Sigma Ampicillin Sigma GenElute Plasmid Mini-Prep Kit Sigma XhoI New England Biolabs XhoI 10X buffer New England Biolabs Agarose EMD % agarose in TAE VDL Accugene 50X TAE Cambrex kb marker Promega X sample loading buffer Promega HiSpeed Plasmid Purification kit Qiagen 3.4. Starting Materials pad5f35 plasmid, 10 μg Transgene cloned into pshuttlex, 10 μg Page 2 of 9

3 4. Procedure CENTER FOR CELL & GENE THERAPY PI-SceI/I-CeuI Digest 4.1. Prepare a 30 μl PI-SceI and I-CeuI double-digest of the recombinant pshuttlex plasmid DNA and the pad5f35 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 3.0 μl Recombinant pshuttlex DNA μg pad5f35 10 μg -- PI-SceI 2.0 μl 2.0 μl I-CeuI 0.5 μl 0.5 μl 10X BSA 3.0 μl 3.0 μl Sterile water Q.S to 30 μl 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 in the bottom of the tube. Page 3 of 9

4 4.18. 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 --- PI-SceI/I-CeuI digest pad5f35 DNA 3 μl 3 μl 5X ligation buffer 2 μl 2 μl DNA ligase 1 μl 1 μl 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. Page 4 of 9

5 4.36. 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 pad5f35 DNA Prepare a 20 μl digest for each experimental and control group as shown in the table below. Reagent SwaI Digest of Ligation Reaction Products Ligation products (from 4.40) 10X SwaI digestion buffer 10X BSA SwaI Restriction Enzyme Total Volume Volume 15 μl 2 μl 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. Page 5 of 9

6 4.55. 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 Stbl2 competent cells with recombinant DNA Thaw one vial of Stbl2 cells on ice Transfer 100 μl Stbl2 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 Stbl2 cells for 25 second at 42 C Immediate transfer to ice and incubate for 2 minutes 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 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 onto the second plate Spread cells across plate with sterile cell spreader Transfer plates to 30ºC 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 each 14 ml tube for each colony selected Inoculate LB 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. Page 6 of 9

7 4.84. Place tubes in a 30ºC shaking incubator for overnight incubation Prepare mini-prep DNA from cultures using the GenElute Plasmid Mini-prep Kit Follow the User s Manual as written except elute the DNA in 50 µl. Restriction Analysis of mini-prep purified DNA Set up the following restriction digest: Test DNA pad5f35 Miniprep purified DNA 20 µl --- pad5f35 DNA µg 10X XhoI buffer 2.5 µl 2.5 µl XhoI 1.0 µl 1.0 µl H 2 O 1.5 µl Q.S. to 25 µl Incubate for 2 hours in a 37ºC water bath During this incubation, prepare a 1.0% agarose gel in TAE After the 2 hour incubation, add 2.5 µl running buffer to each sample Load samples plus 5 µl of the DNA marker on the gel Run gel until bands are clearly resolved If the sequence is available for the pad5f35 + transgene construct, compare the bands on the gel with the predicted sequence If the sequence is not available, compare the bands of the test samples to those of pad5f35. If the test samples show a shift in the ~5kb fragment seen in pad5f35 and only one pattern is present, select one of the test samples with the shifted fragment. If the test samples have more than one pattern, contact the laboratory director to obtain sequence from the investigator before moving on Once the correct plasmid has been identified, inoculate a 3 ml starter culture of LB containing ampicillin with 20 µl of the original culture (4.48) and grow for ~8 hours at 225 rpm at 30 C Dilute the starter culture 1/500 1/1000 in 250 ml LB plus ampicillin. Place culture in a shaking incubator (225 rpm) overnight at 30 C The following day, harvest the bacteria and purify the DNA using the HiSpeed Plasmid Purification Kit. Follow the handbook exactly Determine the concentration of the plasmid using a spectrophotometer Store the DNA at 4 C until use. 5. Data Collection and Management Page 7 of 9

8 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 Director, Vector Production Director, QA/QC Date Issued: 12/11/2006 Replaces VDL Annual Review: 2011 Reviewed without changes Changed and this version archived QA/QC by: Date Issued: 7/30/2011 Replaces VDL Reviewed without changes Changed and this version archived QA/QC by: Date: 2013 Reviewed without changes Changed and this version archived Page 8 of 9

9 QA/QC by: Date: Page 9 of 9