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==TA cloning of the above 8 samples== Step 1. Cloning into pCR2.1. Set up the 10ul ligation reaction as follows: x 9 DNA template 0.5ul 4.5 10x Ligation Buffer 0.5ul 4.5 pCR2.1 vector(25ng/ul) 1ul 9 water 2.5ul 22.5 T4 DNA ligase(4 Weiss Units)0.5ul 4.5 Incubate the ligation reaction at 14C overnight. Step 2.Transforming TOP10 E. coli 1.Equilibrate a water bath to 42C. 2.Bring the SOC medium to room temperature. 3.Take LB plate containing antibotics at 37C for 30 mins. Spread the plate with 80ul of 20mg/ml X_Gal. 4.Centrifuge vials containing the ligation reactions briefly and place them on ice. 5.Thaw on ice one 50ul vial of frozen OneShot TOP10 E coli. 6.Pipette 2ul of each ligation reaction directly into the vial of competent cells and mix by stirring gently with the pipette tip. 7.Incubate the vials on ice for 30 mins. Store the remaining ligation mixtures at -20C. 8.Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials to ice. 9.Add 250ul of room temperature SOC medium into each vial. 10.Shake the vials horizontally at 37C for 1 hour at 225rpm in a shaking incubator. 11.Spread 10ul to 50ul from each transformation bial on LB agar plates containing X_Gal and 50ug/ml of kanamycin. Be sure to plate two different volumes to ensure the at least one plate has well spaced colonies. 12.Incubate plate overnight at 37C.
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