Editing AAV-Production-293T-2 (section)

=== REAGENT SETUP  ===

CRITICAL Unless stated otherwise, reagents can be stored indefinitely at the specified temperature. 

'''CaCl2 (2.0 M)''' Dissolve 29.4 g of CaCl2·6H2O in a final volume of 100 ml dH2O. Sterilize by passing through a 0.22-ÎŒm filter and store in 2-ml aliquots at 4 °C. Cell lysis buffer (for AAV stock preparation) Add 3 ml of 5 M NaCl and 5 ml of 1 M Tris-HCl (pH 8.5) to 80 ml of dH2O. Adjust the pH to 8.5 with NaOH and adjust the volume to 100 ml with dH2O. Sterilize by passing through a 0.22-ÎŒm filter and store at 4 °C. 

'''Church buffer''' Combine 500 ml of 1 M phosphate buffer (pH 7.2), 2 ml of 0.5 M EDTA, 10 g BSA and 70 g SDS in a final volume of 1 liter with dH2O. Store at room temperature (25 °C). 

'''Coomassie blue stain''' Combine 1.5 g Coomassie Brilliant Blue G, 300 ml methanol and 100 ml glacial acetic acid in a final volume of 1 liter with dH2O. Store at room temperature.<br>D10 (DMEM with 10% FBS (vol/vol)) Combine 500 ml of DMEM high glucose with 50 ml of FBS, 5 ml of penicillin/streptomycin and 2.5 ml of amphotericin B (final concentration, 1.25 ÎŒg ml â 1). There is no need to filter-sterilize the solution as all components are sterile. Store at 4 °C and use within 1 month. 

'''Dispase solution''' (0.1% (wt/vol)) Dissolve 100 mg of Dispase in 100 ml of ESC wash medium. Sterilize by passing through a 0.22-ÎŒm filter and store at 4 °C. Use within 1 week. 

'''EDTA solution '''0.5 M (pH 8.0) Dissolve 186.1 g of disodium EDTA·2H2O in 700 ml of H2O. Adjust the pH to 8.0 with NaOH and adjust volume to 1 liter with dH2O. Sterilize by autoclaving and store at room temperature. 

'''ESC medium''' Combine 500 ml of F12/DMEM, 100 ml of KSR, 6 ml of penicillin/streptomycin, 6 ml of sodium pyruvate, 6 ml of nonessential amino acids, 0.6 ml of 0.1 M Î²-mercaptoethanol and 0.3 ml of a 4-ÎŒg/ml bFGF solution. Sterilize by passing through a 0.22-ÎŒm filter and store at 4 °C. Use within 2 weeks. 

'''ESC wash medium''' Add 50 ml of KSR to 500 ml of DMEM high glucose. There is no need to filter sterilize as both components are sterile. Store at 4 °C for up to 2 months. 

'''Gelatin (0.1% (wt/vol))''' Dissolve 5 g of gelatin in 1 liter of dH2O to prepare 0.5% stock solution, sterilize by autoclaving and store at room temperature. Prepare a 0.1% gelatin working solution as a 1:5 dilution of the 0.5% stock in sterile dH2O. Store at room temperature. 

'''Genomic DNA lysis solution''' Combine 1 ml of 1 M Tris-HCl (pH 8.0), 0.2 ml of 0.5 M EDTA (pH 8.0) and 0.1 g of SDS in a final volume of 100 ml with dH2O. Store at room temperature. 

'''HEPES-saline (2Ã)''' Dissolve 8.18 g of NaCl and 5.96 g of HEPES in 400 ml dH2O. Adjust pH to 7.1 with 0.5 N NaOH. Adjust the volume to 500 ml with dH2O. Sterilize by passing through a 0.22-ÎŒm filter and store in aliquots at 4 °C. 

'''Irradiated MEFs''' MEFs are used as feeder cells for the culture of ESCs/iPSCs. DR4 MEFs are used because they are resistant to G418, 6-thioguanine, puromycin and hygromycin, and can be used in experiments that use these selective agents. <br>CRITICAL Prepare the irradiated MEFs in advance as described in Box 1. 

'''KCl (250 mM) ''' Dissolve 1.86 g of KCl in dH2O in a final volume of 1 liter. Sterilize by autoclaving and store at room temperature. 

'''Loading dye (6Ã)''' Combine 5 ml of glycerol, 25 mg of bromophenol blue and 25 mg of xylene cyanol in sterile dH2O in a final volume of 10 ml. Store at 4 °C in 1-ml aliquots. 

'''LB medium (Luria-Bertani medium)''' Dissolve 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl in dH2O in a final volume of 1 liter. Adjust pH to 7.0 with 5 N NaOH. Autoclave and store at room temperature for up to 3 months. 

'''MEF freezing medium ''' To prepare 15 ml of freezing medium, combine 1.5 ml of FBS, 1.5 ml of DMSO and 12 ml of MEF medium. Prepare fresh before use. 

'''MEF medium ''' Mix 500 ml of DMEM high glucose with 50 ml of FBS and 5.6 ml of penicillin/streptomycin. There is no need to filter-sterilize the medium, as all components are sterile. Store at 4 °C and use within 1 month. 

'''MgCl2 (2 M)''' Dissolve 19 g of MgCl2 in dH2O in a final volume of 100 ml. Sterilize by autoclaving and store at room temperature. 

'''MSC medium''' Combine 500 ml of DMEM low glucose with 50 ml of FBS, 5.6 ml of l-glutamine and 5.6 ml of penicillin/streptomycin. There is no need to filter-sterilize the medium as all components are sterile. Store at 4 °C and use within 1 month. 

'''NaCl/PBS-MK buffer (1 M)''' Dissolve 5.84 g of NaCl, 26.3 mg of MgCl2 and 14.91 mg of KCl in 1Ã PBS in a final volume of 100 ml. Sterilize by passing through a 0.22-ÎŒm filter and store at 4 °C. 

'''PBS-MK buffer''' Dissolve 26.3 mg of MgCl2 and 14.91 mg of KCl in 1Ã PBS in a final volume of 100 ml. Sterilize by passing through a 0.22-ÎŒm filter and store at 4 °C. 

'''Phosphate buffer (0.15 M)''' Add 4.95 ml of 1.0 M NaH2PO4 and 10.05 ml of 1.0 M Na2HPO4 to dH2O in a final volume of 100 ml. Sterilize by passing through a 0.22-ÎŒm filter and store in aliquots at 4 °C.<br>Protein precipitation solution Combine 60 ml of 5 M C2H3KO2 and 11.5 ml of glacial acetic acid in a final volume of 100 ml with dH2O. Store at room temperature. 

'''Salmon sperm DNA''' Dissolve salmon sperm DNA in dH2O at a concentration of 10 mg ml â 1. To dissolve the DNA, stir the solution for at least 2â4 h at room temperature. Adjust the concentration of NaCl to 0.1 M and extract the solution once with phenol and once with phenol/chloroform. Recover the aqueous phase and shear the DNA by passing the solution rapidly 12 times through a 17-gauge needle. Precipitate using ethanol, centrifuge and dissolve at a concentration of 10 mg ml â 1. Denature DNA by boiling for 10 min and store at â 20 °C in 1-ml aliquots. Boil for 5 min and snap-chill on ice before use in hybridization. 

'''Super optimal broth (SOB)''' medium Dissolve 20 g of tryptone, 5 g of yeast extract and 0.5 g of NaCl in 900 ml of dH2O. Add 10 ml of 250 mM KCl and adjust pH to 7.0 with 5 N NaOH. Adjust the volume to 1 liter with dH2O and sterilize by autoclaving on a liquid cycle. Store at room temperature for up to 3 months. Add 5 ml of sterile 2 M MgCl2 before use.<br><br>