Transgenic and Gene Targeting Core (TGTC) Preparation of Gene Targeting Vector DNA Purification of plasmid for gene targeting experiments We recommended the Qiagen EndoFree Plasmid Maxi kit for purification of the targeting vector plasmid. In general, avoid overgrowing bacterial cultures containing the targeting vector (≥18 hours of growth at 37 degree centigrade). Growing the scale bacterial cultures used for DNA preparation at 32 degree centigrade often results in better DNA quality. Ensure that ≥90% of the DNA prepared is supercoiled. Linearization of targeting vector:. The targeting vector (gene replacement vectors only) should be linearized using a restriction enzyme site outside the region that is homologous to the target gene. If you have questions about the correct enzyme to use, please contact the Core at The Core requires at least 30ug of linearized targeting vector DNA. The DNA has to be digested to completion. Extraction: Extract the DNA with phenol/chloroform followed by a chloroform extraction to remove proteins. You must use a fresh batch of phenol/chloroform for this purpose. Ensure that the phenol/chloroform mix has a neutral pH. Old phenol/chloroform batches may translate into an unsuccessful gene targeting experiment! Store the purified DNA as a salt/ethanol precipitate at -20 degree centigrade (NaOAc pH 5.2 should be used as the salt). On the day of the electroporation experiment, spin down the DNA precipitate and wash the pellet with 70% ethanol. Briefly air dry the pellet to completely remove traces of alcohol (do not vacuum dry the pellet), and re-suspend the pellet either in TE buffer (10 mM Tris-Cl pH 7.6, 1 mM EDTA) or in PBS at a final concentration of 1mg/ml. Please provide the core with the picture of an agarose gel in which you separated a size marker, undigested targeting vector and linearized targeting vector side-by-side. For further questions please contact (subject: DNA Preparation)
Transgenic and Gene Targeting Core (TGTC) Preparation of Gene Targeting Vector DNA Purification of plasmid for gene targeting experiments We recommended the Qiagen EndoFree Plasmid Maxi kit for purification of the targeting vector plasmid. In general, avoid overgrowing bacterial cultures containing the targeting vector (≥18 hours of growth at 37 degree centigrade). Growing the scale bacterial cultures used for DNA preparation at 32 degree centigrade often results in better DNA quality. Ensure that ≥90% of the DNA prepared is supercoiled. Linearization of targeting vector:. The targeting vector (gene replacement vectors only) should be linearized using a restriction enzyme site outside the region that is homologous to the target gene. If you have questions about the correct enzyme to use, please contact the Core at The Core requires at least 30ug of linearized targeting vector DNA. The DNA has to be digested to completion. Extraction: Extract the DNA with phenol/chloroform followed by a chloroform extraction to remove proteins. You must use a fresh batch of phenol/chloroform for this purpose. Ensure that the phenol/chloroform mix has a neutral pH. Old phenol/chloroform batches may translate into an unsuccessful gene targeting experiment! Store the purified DNA as a salt/ethanol precipitate at -20 degree centigrade (NaOAc pH 5.2 should be used as the salt). On the day of the electroporation experiment, spin down the DNA precipitate and wash the pellet with 70% ethanol. Briefly air dry the pellet to completely remove traces of alcohol (do not vacuum dry the pellet), and re-suspend the pellet either in TE buffer (10 mM Tris-Cl pH 7.6, 1 mM EDTA) or in PBS at a final concentration of 1mg/ml. Please provide the core with the picture of an agarose gel in which you separated a size marker, undigested targeting vector and linearized targeting vector side-by-side. For further questions please contact (subject: DNA Preparation)
info%20DNA%20prep%20gene%20targeting.docx