Jie:LabNotes/CpgSeq/2008-10-16
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Blunt clone and transformation of paralleling sequencing library of BJ and Hybrid_2 constructed on 2008-09-12[edit]
refer to LabNotes on [1]
Cloning into pCR2.1.
Set up the 10ul ligation reaction as follows: PCR product of sequencing library 0.5ul 10x Ligation Buffer 0.5ul pCR2.1 vector(25ng/ul) 0.5ul water 3ul T4 DNA ligase(4 Weiss Units) 0.5ul
Incubate the ligation reaction at 16C 1h.
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 45 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.
PCR
set up the PCR reaction system. reaction system x16 ddH2O 11ul 176ul Taq master mix(2x) 15ul 240ul M13 Forward primer (10uM) 2ul 32ul M13 Reverse primer (10mM) 2ul 32ul Clone template 1ul 1ul each Pick up 8 clones as the template.
94C 3min -> 30 cycles of(94C 45sec -> 55C 30sec ->72C 45 sec)-> 72C 3mins ->4C