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Gene Function Analysis Using the Chicken B-Cell Line 2035. 500 µL of the upper phase are transferred to a new tube containing 500 µL chloroform.6. The solution is mixed by inverting and centrifuged at 13,000 rpm and 4°C for 5min.7. 500 µL of the upper phase are transferred to a new tube containing 1µL RNAse A.8. The RNA is digested for 2 h at 37°C.9. The reaction is stopped by the addition of 50 µL of 0.5 M EDTA pH 8.0 and mixingby gently inverting.10. Perform a quick spin (the DNA can be stored at this point at 4°C until dialysis).11. The DNA is loaded into a dialysis membrane and dialyzed against ice cold 1X TEat 4°C while mixing. The 1X TE is changed after 2 h and again after 2–4 h followedby overnight dialysis.Day 3: transfer the DNA from the membrane to a clean tube and measure theOD260 to obtain the concentration.3.1.2. Amplification of the Target Arm SequencesThe 5′ and 3′ ends flanking the knockout region of the target gene are amplifiedby PCR. Use the Expand Long Template PCR System (Roche AppliedScience) with cresol red, 10 mM dNTP, forward primer (25 pM), and reverseprimer (25 pM) in the standardized amplification reaction mixture (target armsPCR). The mixture is evenly divided among three PCR tubes.PCR program:93°C 2 min93°C 10 s ⎫⎪ 35 cycles65°C 30 s ⎬68°C 5 min*⎪ ∗time increases: 20 s each cycle.⎭68° C 7 min4C ° ∞Five microliter of each sample are checked and confirmed through gel electrophoresis.The selected samples are combined for PCR purification throughethanol precipitation or a commercially available kit of one’s choice. If at firstthe PCR fails, repeat once using half the amount of primer and the addition of1 µL DMSO (see Note 2).3.1.3. Preparing Arms and Vector for CloningThe target arm sequences are now cloned into the vector pKS using chemicalor electro competent Escherichia coli cells. Sequentially, the 5′ and 3′arms are cloned into the appropriately restricted vector. In the final step, themarker cassettes are cloned into the targeting vector between the two armsusing the BamHI and/or BglII site(s). The targeting of the two alleles of agene is accomplished by using two different resistance marker cassettes that