Molecular Cloning, Product Brochure

Transcription

1 , Product Brochure Interest in any of the products, request or order them at Bio-Connect. Bio-Connect B.V. T NL +31 (0) T BE +32 (0) Begonialaan 3a F NL +31 (0) F BE +32 (0) TE Huissen E info@bio-connect.nl The Netherlands W

2 Enzymes 2-1 TA Cloning Kits 2-4

3 Enzymes T4 DNA Ligase 2-1 T4 DNA Ligase T4 DNA ligase catalyzes the linkage of juxtaposed 5 -phosphate and 3 -hydroxyl termini in duplex DNA or RNA through the formation of a phosphodiester bond. The enzyme will join cohesive and blunt end termini and also will repair single stranded nicks in duplex DNA, RNA as well as DNA/RNA hybrids. Applications - Joining double-stranded nucleic acid fragments with cohesive or blunt ends. - Repair nicks in duplex DNA, RNA, and DNA/RNA hybrids. Concentration: 10 units/µl Weiss unit is the amount of T4 DNA Ligase needed to catalyze the ligation of greater than 95% of 1µg of λ/hind III fragments at 16 C in 20 minutes. Colony Count Time (min) Accelerated Ligation vs Normal Ligation: Ligation proceeded at a 1:1 insert:vector ratio with 1 unit of ligase to specified time points at room-temperature. Ligation products were transformed into DH5a (ABP-CE-CC02005) competent E. coli cells, grown overnight on LB-amp plates at 37 C. High concentration T4 DNA Ligase with 10X Buffer ABP-CE-T4L10X1 100 U ABP-CE-T4L10X5 500 U High concentration T4 DNA Ligase with 2X Buffer ABP-CE-T4L2X U ABP-CE-T4L2X U T4 DNA Polymerase Allele Enzymes offers an affinity purified, highly active, recombinant T4 DNA Polymerase that in the presence of template and primer, catalyzes the synthesis of DNA in the 5 -> 3 direction. The enzyme has a strong 3 -> 5 exonuclease activity; it has no exonuclease activity in the 5 -> 3 direction. It contains no other DNAse or RNAse activities.

4 2-2 Enzymes T4 DNA Polymerase Applications - Fill-in 3 recessive ends of double-stranded DNA - Removal of 3 overhangs to form blunt ends - Probe labelling by replacement synthesis - Strand synthesis during mutagenesis Concentration: 5 units/µl One unit of T4 DNA polymerase is the amount of enzyme activity that incorporates 10 nmol of total nucleotides into acid-insoluble material in 30 minutes at 37 C. T4 DNA Polymerase ABP-CE-T4PO U ABP-CE-T4PO U T4 Polynucleotide Kinase Allele Biotech offers a highly purified recombinant T4 Polynucleotide Kinase that is especially suitable for sensitive probe labeling purposes. T4 Polynucleotide Kinase catalyzes the transfer of the γ-phosphate of ATP to the 5 -hydroxyl terminus of DNA and RNA molecules. Applications - End labeling DNA or RNA for use as probes - Hydrolysis of 3 -phosphoryl group - Phosphorylating oligonucleotides for use in subsequent ligations Concentration: 10 units/µl One unit is the amount of enzyme necessary to catalyze the transfer of 1nmol of phosphate to the 5 -hydroxyl end of a polynucleotide from the [γ P32] -phosphate of ATP in 30 minutes at 37 C. T4 Polynucleotide Kinase ABP-CE-TPK100U 100 U ABP-CE-TPK U

5 Enzymes Clean Cut Protease 2-3 Clean-Cut Protease Clean-CutTM protease is a viral 3C family protease cloned from a novel strain of HCV by Allele Biotech. The enzyme specifically recognizes a cleavage site of ELWSQ/X (X can be any small amino acid). Features - Cleaves at a highly specific recognition site. Unlike thrombin or factor X, Clean-Cut protease does not generate non-specific product bands in most cases, even after long incubation. - Functions well at both room temperature and 4 C, and does not require specific buffers. The digestion reaction can be simply carried out in PBS or Tris-based elution buffer, or even on beads. - Reactions only take one hour for most commonly used purposes Concentration: 10 units/µl Each unit is defined as the amount of enzyme necessary to cleave ~ 50 µg target protein in one hour at room temperature. Typical specific activity: units/ µg of protease. Above Image: CleanCutTM Protease digestion of a His-tag fusion protein of 25kD bound to Ni-beads. Units and incubation time are indicated. At the end of the reaction, the beads were washed once with PBS. A portion was run on 14% SDS-PAGE. Control lane was not treated; the bound protein was eluted by 500mM imidazol containing elution buffer. CleanCut Protease ABP-CE-CCP U

6 2-4 Cloning TA Cloning TA Cloning Vector The Allele TA Vector is a high quality, cloning vector used to facilitate the cloning of PCR products. This vector has been linearized and is flanked by many recognition sites for restriction enzymes. Allele TA Vector possesses single 3 thymidine overhangs at the insertion site, which improve ligation efficiency of PCR products into the vector. The 3 T overhang prevents recirculization of the vector and provides matching nucleotides to the protruding 3 deoxyadenosine on the PCR product. Allele TA Vector also facilitates the analyses of the positive clones just by a single digest for 15min~1hour (Hind III or Kpn I, see the MCS map). Features - Suitable for cloning PCR products - Low background - Blue/White Screening - Rapid Screening General Vector Data Vector size: 2708 bp Cloning Site: 437 Primer Binding Site M13F: TGTAAAACGACGGCCAGT ( ) M13R: CAGGAAACAGCTATGAC ( ) Suggested protocol for DNA ligation - Allele TA vector (10ng/ul): 1ul - PCR product: 1-5ul - Ligation buffer (10x): 1ul - T4 DNA ligase: 0.5ul - Distilled water: add to 10ul Incubate at 4-16 C overnight (rapid ligation in room temperature is not recommend) Transformation Suitable for a quick 3 min protocol using Allele s DH5α Extreme Efficiency Competent Cells(ABP-CE-CC02005) Select and Analyze - Blue/White Screening is recommended - Analyze the positive clone: digest by Hind III, Kpn I or any other applicable enzyme TA Cloning Vector ABP-CE-TAVEC RXNs TA Cloning Kit ABP-CE-TAKIT RXNs

7 PCR Cloning TA Cloning 2-5 Multiple Cloning Site Sequence TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA 60 CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG 120 TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC 180 ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC 240 ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT 300 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT 360 TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT ACCCGGGGAT 420 CCTCTAGATC CAAGCTT AGG CCTGGTACCG GTCGACCTGC AGGCATGCAA GCTTGGCGTA 480 Cloning site ATCATGGTCA TAGCTGTTTC CTGTGTGAAA TTGTTATCCG CTCACAATTC CACACAACAT 540 ACGAGCCGGA AGCATAAAGT GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT AACTCACATT 600 AATTGCGTTG CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC AGCTGCATTA 660 ATGAATCGGC CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCTCTT CCGCTTCCTC 720 GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG CTCACTCAAA 780 GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA 840 AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT 900 CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC 960 AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC 1020 GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC 1080 TCATAGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG 1140 TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA 1200 GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG 1260 CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA 1320 CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT TCGGAAAAAG 1380 AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT TTTTTGTTTG 1440 CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA TCTTTTCTAC 1500 GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC 1560 AAAAAGGATC TTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT CAATCTAAAG 1620 TATATATGAG TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG CACCTATCTC 1680 AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT AGATAACTAC 1740 GATACGGGAG GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG ACCCACGCTC 1800 ACCGGCTCCA GATTTATCAG CAATAAACCA GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG 1860 TCCTGCAACT TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG CTAGAGTAAG 1920 TAGTTCGCCA GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTACAGGCA TCGTGGTGTC 1980 ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA GGCGAGTTAC 2040 ATGATCCCCC ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG 2100 AAGTAAGTTG GCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA ATTCTCTTAC 2160 TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA AGTCATTCTG 2220 AGAATAGTGT ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAATACGGG ATAATACCGC 2280 GCCACATAGC AGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG GGCGAAAACT 2340 CTCAAGGATC TTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG CACCCAACTG 2400 ATCTTCAGCA TCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG GAAGGCAAAA 2460 TGCCGCAAAA AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC TCTTCCTTTT 2520 TCAATATTAT TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG 2580 TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG TGCCACCTGA 2640 CGTCTAAGAA ACCATTATTA TCATGACATT AACCTATAAA AATAGGCGTA TCACGAGGCC 2700 CTTTCGTC

8 2-6 Notes