t4 dna polymerase  (New England Biolabs)


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    Structured Review

    New England Biolabs t4 dna polymerase
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 344 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    In Vitro:

    Article Title: Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.
    Article Snippet: 98 Double-strand DNAs were created by T4 DNA polymerase (New England Biolabs Inc.) 99 after annealing both gene-specific and constant oligonucleotides. .. In vitro transcription 100 was employed for gRNA synthesis using HiScribeâ„¢ SP6 RNA Synthesis Kit (New 101 England Biolabs Inc.).

    Synthesized:

    Article Title: Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.
    Article Snippet: All the oligonucleotides 97 (Supplementary Table 1) were synthesized by IDT (Integrated DNA Technologies). .. 98 Double-strand DNAs were created by T4 DNA polymerase (New England Biolabs Inc.) 99 after annealing both gene-specific and constant oligonucleotides.

    Isolation:

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens
    Article Snippet: EcoP15 I-generated sticky ends were end-filled with T4 DNA polymerase by adding 1.5 μL T4 DNA polymerase (4.5 U), 1.5 μL NEBuffer-2 (NEB), and 2 μL dNTP (1 mM, Sangon) to a total 15-μL reaction and incubated at 37°C for 15 min. Fragments of appropriate sizes (63-67 bp) were purified from 20% Tris-Borate-Ethylene Diamine Tetraacetic Acid (TBE) polyacrylamide gels using QIAEX II Gel Extraction Kit (Qiagen) and eluted with ddH2 O. .. Next, 2.5 μL of DNA isolated by PAGE from step 4 was supplemented with 0.5 μL 10× ligation reaction buffer [500 mM Tris-HCl (pH 7.5, 25°C), 100 mM MgCl2 , 100 mM DTT, 25 μg/mL BSA]; 1 μL loop-2 linker (20 mM, Sigma-Aldrich); 0.5 μL ATP (10 mM, Sigma-Aldrich); 0.5 μL T4 DNA Ligase (NEB); and 0.5 μL ddH2 O.

    Ligation:

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens
    Article Snippet: Paragraph title: Step 5. End-filling and loop-2 linker ligation ... EcoP15 I-generated sticky ends were end-filled with T4 DNA polymerase by adding 1.5 μL T4 DNA polymerase (4.5 U), 1.5 μL NEBuffer-2 (NEB), and 2 μL dNTP (1 mM, Sangon) to a total 15-μL reaction and incubated at 37°C for 15 min. Fragments of appropriate sizes (63-67 bp) were purified from 20% Tris-Borate-Ethylene Diamine Tetraacetic Acid (TBE) polyacrylamide gels using QIAEX II Gel Extraction Kit (Qiagen) and eluted with ddH2 O.

    Incubation:

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens
    Article Snippet: .. EcoP15 I-generated sticky ends were end-filled with T4 DNA polymerase by adding 1.5 μL T4 DNA polymerase (4.5 U), 1.5 μL NEBuffer-2 (NEB), and 2 μL dNTP (1 mM, Sangon) to a total 15-μL reaction and incubated at 37°C for 15 min. Fragments of appropriate sizes (63-67 bp) were purified from 20% Tris-Borate-Ethylene Diamine Tetraacetic Acid (TBE) polyacrylamide gels using QIAEX II Gel Extraction Kit (Qiagen) and eluted with ddH2 O. .. The polyacrylamide gel electrophoresis (PAGE)–purified blunt-ended DNA fragments were ligated to a loop-2 linker containing a Fok I recognition sequence (CATCC).

    Article Title: Enzymatic Synthesis of Designer DNA Using Cyclic Reversible Termination and a Universal Template.
    Article Snippet: Phosphoramidite chemistry remains the industry standard for DNA synthesis despite significant limitations on the length and yield of the oligonucleotide, time restrictions, and hazardous waste production. .. Phosphoramidite chemistry remains the industry standard for DNA synthesis despite significant limitations on the length and yield of the oligonucleotide, time restrictions, and hazardous waste production.

    Purification:

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens
    Article Snippet: .. EcoP15 I-generated sticky ends were end-filled with T4 DNA polymerase by adding 1.5 μL T4 DNA polymerase (4.5 U), 1.5 μL NEBuffer-2 (NEB), and 2 μL dNTP (1 mM, Sangon) to a total 15-μL reaction and incubated at 37°C for 15 min. Fragments of appropriate sizes (63-67 bp) were purified from 20% Tris-Borate-Ethylene Diamine Tetraacetic Acid (TBE) polyacrylamide gels using QIAEX II Gel Extraction Kit (Qiagen) and eluted with ddH2 O. .. The polyacrylamide gel electrophoresis (PAGE)–purified blunt-ended DNA fragments were ligated to a loop-2 linker containing a Fok I recognition sequence (CATCC).

    Article Title: Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.
    Article Snippet: 98 Double-strand DNAs were created by T4 DNA polymerase (New England Biolabs Inc.) 99 after annealing both gene-specific and constant oligonucleotides. .. All the gRNAs were then purified using a Zymo RNA concentrator-5 kit 103 (Zymo Research) or MEGAclear™ Transcription Clean-Up Kit (TheromFisher) following 104 manufacturer’s instructions.

    Generated:

    Article Title: Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.
    Article Snippet: 98 Double-strand DNAs were created by T4 DNA polymerase (New England Biolabs Inc.) 99 after annealing both gene-specific and constant oligonucleotides. .. Usually, > 10µg RNA can be generated per reaction within 2-4 102 hours at 37 ºC.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens
    Article Snippet: EcoP15 I-generated sticky ends were end-filled with T4 DNA polymerase by adding 1.5 μL T4 DNA polymerase (4.5 U), 1.5 μL NEBuffer-2 (NEB), and 2 μL dNTP (1 mM, Sangon) to a total 15-μL reaction and incubated at 37°C for 15 min. Fragments of appropriate sizes (63-67 bp) were purified from 20% Tris-Borate-Ethylene Diamine Tetraacetic Acid (TBE) polyacrylamide gels using QIAEX II Gel Extraction Kit (Qiagen) and eluted with ddH2 O. .. The polyacrylamide gel electrophoresis (PAGE)–purified blunt-ended DNA fragments were ligated to a loop-2 linker containing a Fok I recognition sequence (CATCC).

    CRISPR:

    Article Title: Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.
    Article Snippet: For gene-specific oligonucleotides, SP6 promoter 94 sequence was added before the CRISPR RNA sequence followed by an overlap 95 adaptor, which is complementary to the 5’ end of an 80bp constant oligonucleotide 96 5 according to the published protocol (Gagnon et al. 2014). .. 98 Double-strand DNAs were created by T4 DNA polymerase (New England Biolabs Inc.) 99 after annealing both gene-specific and constant oligonucleotides.

    Sequencing:

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens
    Article Snippet: EcoP15 I-generated sticky ends were end-filled with T4 DNA polymerase by adding 1.5 μL T4 DNA polymerase (4.5 U), 1.5 μL NEBuffer-2 (NEB), and 2 μL dNTP (1 mM, Sangon) to a total 15-μL reaction and incubated at 37°C for 15 min. Fragments of appropriate sizes (63-67 bp) were purified from 20% Tris-Borate-Ethylene Diamine Tetraacetic Acid (TBE) polyacrylamide gels using QIAEX II Gel Extraction Kit (Qiagen) and eluted with ddH2 O. .. The polyacrylamide gel electrophoresis (PAGE)–purified blunt-ended DNA fragments were ligated to a loop-2 linker containing a Fok I recognition sequence (CATCC).

    Article Title: Enzymatic Synthesis of Designer DNA Using Cyclic Reversible Termination and a Universal Template.
    Article Snippet: Phosphoramidite chemistry remains the industry standard for DNA synthesis despite significant limitations on the length and yield of the oligonucleotide, time restrictions, and hazardous waste production. .. Phosphoramidite chemistry remains the industry standard for DNA synthesis despite significant limitations on the length and yield of the oligonucleotide, time restrictions, and hazardous waste production.

    Article Title: Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.
    Article Snippet: For gene-specific oligonucleotides, SP6 promoter 94 sequence was added before the CRISPR RNA sequence followed by an overlap 95 adaptor, which is complementary to the 5’ end of an 80bp constant oligonucleotide 96 5 according to the published protocol (Gagnon et al. 2014). .. 98 Double-strand DNAs were created by T4 DNA polymerase (New England Biolabs Inc.) 99 after annealing both gene-specific and constant oligonucleotides.

    Gel Extraction:

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens
    Article Snippet: .. EcoP15 I-generated sticky ends were end-filled with T4 DNA polymerase by adding 1.5 μL T4 DNA polymerase (4.5 U), 1.5 μL NEBuffer-2 (NEB), and 2 μL dNTP (1 mM, Sangon) to a total 15-μL reaction and incubated at 37°C for 15 min. Fragments of appropriate sizes (63-67 bp) were purified from 20% Tris-Borate-Ethylene Diamine Tetraacetic Acid (TBE) polyacrylamide gels using QIAEX II Gel Extraction Kit (Qiagen) and eluted with ddH2 O. .. The polyacrylamide gel electrophoresis (PAGE)–purified blunt-ended DNA fragments were ligated to a loop-2 linker containing a Fok I recognition sequence (CATCC).

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    New England Biolabs dnase i buffer
    The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using <t>DNase</t> I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.
    Dnase I Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i buffer/product/New England Biolabs
    Average 90 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    dnase i buffer - by Bioz Stars, 2020-04
    90/100 stars
      Buy from Supplier

    99
    New England Biolabs t4 dna ligase
    Schematic illustration of the multiple patch cloning procedure. DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and <t>T4</t> DNA ligase.
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3478 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/New England Biolabs
    Average 99 stars, based on 3478 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

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    The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.

    Journal: Nucleic Acids Research

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA

    doi: 10.1093/nar/gkr051

    Figure Lengend Snippet: The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.

    Article Snippet: Pellets were suspended in 5 µl DNase I buffer and 0.2 U DNase I (NEB) and 0.2 U snake venom phosphodiesterase (Worthington) was added to the reactions.

    Techniques: Incubation, Labeling, Thin Layer Chromatography, High Performance Liquid Chromatography, Mass Spectrometry, Polymerase Chain Reaction, Liquid Chromatography with Mass Spectroscopy, Modification

    Schematic illustration of the multiple patch cloning procedure. DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.

    Journal: BMC Biotechnology

    Article Title: Patch cloning method for multiple site-directed and saturation mutagenesis

    doi: 10.1186/1472-6750-13-91

    Figure Lengend Snippet: Schematic illustration of the multiple patch cloning procedure. DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.

    Article Snippet: The MUPAC enzyme stock solution was prepared by mixing 10 μL of T5 exonuclease (10 U/μL; New England Biolabs), 1 μL of Klenow fragment exo– (5 U/μL; New England Biolabs), 2.5 μL of T4 DNA ligase (400 U/μL; New England Biolabs), and 11.5 μL of the storage buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, and 0.1% Triton X-100).

    Techniques: Clone Assay, Amplification, Polymerase Chain Reaction, Plasmid Preparation

    Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.

    Journal: Nucleic Acids Research

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries

    doi: 10.1093/nar/gkv724

    Figure Lengend Snippet: Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.

    Article Snippet: Because our approach relies on T4 DNA Ligase, which is sensitive to base-pair mismatches and gaps , these variations can adversely affect the efficacy of the blocking (Supplementary Figure S4).

    Techniques: Modification, Sequencing, Blocking Assay, Polymerase Chain Reaction, Ligation, Generated, Titration

    Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer

    Journal: Journal of Clinical Microbiology

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    doi: 10.1128/JCM.02340-10

    Figure Lengend Snippet: Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer

    Article Snippet: To circularize the HBV genome with minimum intermolecular ligation, 1.5 μg of the digest or gel-purified HBV DNA was ligated at 16°C overnight with 1,600 U (4 μl) of T4 DNA ligase (New England BioLabs) in a total volume of 1.5 ml.

    Techniques: Transfection

    Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and

    Journal: Journal of Clinical Microbiology

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    doi: 10.1128/JCM.02340-10

    Figure Lengend Snippet: Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and

    Article Snippet: To circularize the HBV genome with minimum intermolecular ligation, 1.5 μg of the digest or gel-purified HBV DNA was ligated at 16°C overnight with 1,600 U (4 μl) of T4 DNA ligase (New England BioLabs) in a total volume of 1.5 ml.

    Techniques: Functional Assay, Polymerase Chain Reaction, Clone Assay, Transfection