PCR Product T/A Cloning Vector Kit

PCR Product T/A Cloning Vector Kit Cat. No CV-­‐M-­‐001-­‐20 Store at -­‐20°C Components pUCm-­‐T vector (50ng/μl) 1 μg 10 x Ligation Buffer 50 μl 50% PEG 4000 50 μl T4 DNA ligase, 5U/μl 100 U Sterilized ddH2O 1 ml Protocol 1 Description The MOLEQULE-­‐ON PCR Product T/A Cloning Vector Kit is suitable for cloning of PCR products with additional A at 3’ end. The special ligation system provided by the kit enables the whole process to finish ligation in 1-­‐2 hour. pUCm-­‐T vector is designed to simply clone the PCR products. Many thermal stable DNA polymerase, such as Taq, Tth DNA produce additional A at 3’ end, which could be easily ligated to T vector with additional T. pUCm-­‐T is a novel pUC derivative T vector, possesses multiple restriction sits with most of them having single site only and adjusted β-­‐galactose reading frame make it easy for screening target clone through blue and white colonies. Specially designed two PstI sites beside inserted fragments make it easy for screening target clone by PstI digestion, at same time inserted fragments also could be screened by cheap and efficient restriction enzymes such as EcoRI and HindIII double digestion. Inserted fragments could be sequenced by using universal primers M13 and T7 promoter primer. In pUCm-­‐T, in vitro transcription could be processed through site of T7 RNA polymerase promoter. Preparation of Ligation Reaction Before ligating the DNA fragment into cloning vector, it is recommended to purify or gel extract the PCR product. Moreover, if plasmids were used as template, it is necessary to purify the PCR product, because plasmid template could form white colony after transformation. PCR product purification kit (PP-­‐M-­‐001-­‐50) and gel extraction kit (GE-­‐M-­‐001-­‐50) of MOLEQULE-­‐ON could recover >60 bp DNA fragment efficiently. PCR products amplified by Taq, Tth, AmpliTaq, KlenTaq DNA polymerase bear additional A at 3’end. Taq DNA polymerase co-­‐
amplify with Pfu, Pwo, Tli or Deep vent DNA polymerase which posses 3→5 exonuclease activity may bear a additional A at 3’ end. PCR products with additional A at 3’ end could be ligated to pUCm-­‐T vector. PCR products amplified by DNA polymerases possess 3’→5 exonuclease activity are blunt end, cloning of this kind of fragments need to add additional A to blunt end. A-­‐
Tailing Kit (AT-­‐M-­‐001-­‐40) of MOLEQULE-­‐ON could be efficiently used to add additional A to blunt 3’ end PCR product. www.molequle-­‐on.com Ligation Reaction For a standard 10μl reaction mixture, add the following components one by one carefully. Usually it is not necessary to quantify the PCR products, however, 0.2 pmol of PCR product is recommended. Add ddH2O to make the final 10μl volume. 10 x ligation Buffer 1 μl 50% PEG 4000 1 μl pUCm-­‐T Vector 1 μl Purified PCR Product X μl T4 DNA ligase 1 μl Sterilized ddH2O Y μl Total Volume 10 μl Incubate the ligation mixture for 1 hour to overnight at 16~23°C. Usually 1 hour is sufficient for normal experiment. Transformation The competent cells of E. coli can be prepared by using MOLEQULE-­‐ON Competent Cell Preparation Kit (CP-­‐M-­‐001-­‐25; NO HEAT SHOCK REQUIRED). 1. Put fresh or frozen 100μl of E. coli competent cell thawed on ice, gently mix well after completely thawed. 2. Add 5μl of ligation mixture, gently mix well. Keep on ice for 30 min. 3. Heat shock at 42°C on water bath for 1 min. Keep on ice for 2 min. 4. Add 400μl SOC culture, shake on 200-­‐250 rpm at 37°C for 1 hour. 5. Centrifuge at 4000rpm for 5 min, discard 300μl supernatant using pipette, resuspend/shake the pelleted cells with rest of culture. 6. Plate bacteria at Amp+ (100 μg/ml) plates, which had been plated with 20μl of 100mM IPTG and 100μl of 20mg/ml X-­‐gal. Note: Efficiency of ligation and transformation depends on quantity of bacteria used. 7. Incubate the inoculated plate overnight at 37°C. Screening Screened transformants by blue/white colony When foreign DNA fragment cloned into pUCm-­‐T vector, reading frame of LacZ gene coding sequence changed and its product become inactive. Recombinant clone on X-­‐gal/IPTG plate display white colony, at the same time non-­‐recombinant display blue colony. For the identification of transformants, select white colony on IPTG/X-­‐gal plate, transfer colony to Amp+ liquid media under sterile conditions and culture overnight at 37°C. www.molequle-­‐on.com Identification of Transformants 1. Extract plasmid from liquid culture of white colony, digest plasmid by PstI or other suitable restriction enzymes, and check the size of inserted fragment in order to decide whether the clone contains target fragment. 2. Colony PCR can be performed in order to confirm the presence of desired sequence in white recombinant colony by using M13 universal or gene specific primer. 3. The sequence of target clone can be determined by M13 universal primer or other suitable primer to further confirm the inserted fragment. Map of pUCm-­‐T Vector The complete sequence of pUCm-­‐T vector is same as pUC19 (GenBank Accession Number M77789) except difference at multiple cloning sites. www.molequle-­‐on.com For Research Use Only