T载体说明书
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1、ReagentsStandardReactionPositive controlBackground controlSolution I5pl5pl5plpUC-T Easy Vector (50ng)1pl1pl1plPCR productXpl一Control Insert DNA2pl一Deionized water to a final volume of10pl10pl10pl3. Set up ligation reactions as described below. Vortex the Solution I vigorously before each use. Use 0.
2、5ml tubes known to have low DNA Binding capacity.Cloning PCR Products with pUC-T Easy Vectors .Product contentsProduct No.T1101-1T1101-2T1101-3SIZE1.0ug5.0ug10.0ugSolution I50ul250ul500ulControl Insert10ul50ul100ul18T Vector10ul50ul100ulInstruction Booklet1112. Briefly centrifuge the pUC-T Easy Vect
3、or and Control Insert DNA tubes to collect contents at the bottom of the tube.4.Mix the reactions by pipetting. Incubate the reactions 1 hour at room temperature. Alternatively, incubate the reactions overnight at 4C for the maximum number of transformants.Transformation of DH5- a or Other Strain Hi
4、gh Efficiency Competent Cells1. Prepare LB/ampicillin/IPTG/X-Gal plates.2. Centrifuge the ligation reactions briefly. Add 2pl of each ligation reaction to asterile 1.5ml tube on ice. Prepare a control tube with 0.1ng of uncut plasmid.3. Place the DH5-a High Efficie ncy Compete nt Cells in an ice bat
5、h un til just thawed (5 minu tes). Mix cells by gently_ flick ing the tube.4. Carefully transfer 50pl of cells to the ligation reaction tubes from Step 2. Use 100pl of cells for the uncut DNA control tube . Gently flick the tubes and Incubate on ice for 20 minutes.5. Heat-shock the cells for 45-50 s
6、eco nds in a water bath at exactly 42C. DO NOT SHAKE. Immediately return the tubes to ice for 2 minutes.Thaw competentcells on ice. Add 50plcells to 2pl of theligation reaction.Incubate on ice forIncubate on icefor 2 minutes.Add SOC medium.Incubate for 1.5 hours at37 Cwith shaking.Plate. Select whit
7、ecolonies.6. Add 950pl room temperature SOC medium to the ligati on reacti on tran sformatio nsand 900pl to the uncut DNA control tube. Incubate for 1.5 hours at 37C with shaking (150rpm)7. Plate 100pl of each tran sformatio n culture onto duplicate LB/ampicilli n/IPTG/XGal plates. For the uncut DNA
8、 control, a 1:10 dilution with SOC is recommended.8.Incubate plates overnight at 37C. Select white colonies.EcaBestTM Seque ncing Primer RV-MGAGCGGATAACAATTTCACACAGGEcoR ISac I Kpn II II II15-GAGCGGATAACAATTTCACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCAGGGC T-BamH I Xba I EcoR VI ii iiSal GATCTG
9、GGGATCCTCTAGAGATATCGTCGACCTTctagacccctaggagatctctatagcagctgga刊 nd III|ACCTCGCCTATTGTTAAAGTGTCCTTTGTCGATACTGGTACTAATGCTTAAGCTCGAGCCATGGGGTCCCGT-CI one SiteGCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCG-3CGTCCGTACGTTCGAACCGTGACCGGCAGCAAAATGTTGCAGCACTGACCCTTTTGGGACCGCCAGCACTGACCCTTTTGGGACCGCBcaBestTM Sequencing Primer M13-47
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