Immunoglobulin variable heavy and light chain variable regions (VH and VL) were RT-PCR amplified using AmpliTaq360 Master Mix (Applied Biosystems) with conditions previously described [55 (link)]. PCR products were purified (Qiagen, Valencia, CA) and sequenced with a BigDye Sequencing kit (Applied Biosystems) on an ABI 3700 sequencer. VH and VL chain gene rearrangements, clonal relatedness, UCA and intermediate ancestor (IA) inferences were made using Cloanalyst [56 (link)].
Amplitaq 360 master mix
Manufactured by Thermo Fisher Scientific
AmpliTaq 360 Master Mix is a pre-optimized PCR reagent solution that contains AmpliTaq 360 DNA Polymerase, dNTPs, and buffer components. It is designed to provide robust and reliable amplification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Topo ta cloning kit, by Thermo Fisher Scientific (2 mentions)
3130xl genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Exonuclease 1 and alkaline phosphatase shrimp, by Takara Bio (2 mentions)
Bigdye terminator cycle sequencing kit v3, by Thermo Fisher Scientific (2 mentions)
Gc enhancer, by Thermo Fisher Scientific (1 mentions)
Seqscape version 2, by Thermo Fisher Scientific (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Abi bigdye terminator chemistry, by Thermo Fisher Scientific (1 mentions)
6 protocols using amplitaq 360 master mix
1
Amplification and Sequencing of Immunoglobulin Chains
2
Cloning and Sequencing of PCR Products
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The PCR products were cloned with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A total of 96 colonies were randomly selected from each clone library for sequencing analysis. The partial fragments of the cloning vectors (pCR 4) containing inserted PCR products were amplified with AmpliTaq 360 Master Mix, GC Enhancer and Pre-Seq primer set (F; 5’-GTTTTCCCAGTCACGACG-3’ and R; 5’-CAGGGAACAGCTATGAC-3’, Applied Biosystems). After the primers and deoxyribonucleotided triphosphate were eliminated from PCR mixture with an Exonuclease I and Alkaline Phosphatase (Shrimp) (TaKaRa Bio Inc., Otsu, Shiga, Japan) according to the manufacturer’s instructions, a 1 μL aliquot was used as a template for the sequencing reaction. The sequencing reactions were accomplished with primers M13 F and BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems). The nucleic acid sequences were determined on a 3130xl Genetic Analyzer (Applied Biosystems).
3
Cloning and Sequencing of PCR Amplicons
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The amplified PCR products were cloned into Escherichia coli using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA), and then the nucleotide sequences of 96 randomly selected colonies were determined from each clone library for a sequencing analysis. Subsequently, we amplified the partial fragments of the cloning vectors (pCR 4) with AmpliTaq 360 Master Mix, GC Enhancer and a Pre-Seq primer set (F; 5′-GTTTTCCCAGTCACGACG-3′ and R; 5′- CAGGGAACAGCTATGAC-3′; Applied Biosystems). An Exonuclease I and Alkaline Phosphatase (Shrimp) (TaKaRa Bio Inc., Otsu, Shiga, Japan) was used to eliminate the primers and deoxyribonucleotided triphosphate from the PCR mixture in a clean-up process. From that, a 1 µl aliquot was used as a template for the sequencing reaction to perform with primers M13 F and the BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems). The nucleic acid sequences were determined on a 3130xl Genetic Analyzer (Applied Biosystems) as the final of the process.
4
Splinkerette-PCR for Transgene Integration Site
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To isolate the insertion site of the CM32 transgene, splinkerette-PCR and sequencing were
performed, according to a previously described procedure [24 (link)] with a minor modification. Briefly, tail DNA from the transgenic mouse was
digested with PstI and used for adaptor ligation. AmpliTaq360 master mix
(Life Technologies) was used for PCR. The oligonucleotides for a splinkerette adaptor are
5’-CGAAGAGTAACCGTTGCTAGGAGAGACCGTGGCTGAATGAGACTGGTGTCGACACTAGTGGtgca-3’ and
5’-CCACTAGTGTCGACACCAGTCTCTAATTTTTTTTTTCAAAAAAA-3’. Primers used for splinkerette-PCR are
as follows: 5’-CGAAGAGTAACCGTTGCTAGGAGAGACC-3’ (Splink1) and
5’-CATAATGCCAGGCGGGCCATTTACC-3’ (CMVIE-4R) for primary PCR amplification; and
5’-GTGGCTGAATGAGACTGGTGTCGAC-3’ (Splink2) and 5’-GGGCGTACTTGGCATATGATACACTTGATG-3’
(CMVIE-5R) for secondary PCR amplification.
The 3’ portion of the integrated transgene was PCR-amplified using CM32 Tg genomic DNA
and the primers 5’-TGCTCCTGGAGATGTTGGATG-3’ and 5’-TCAGTGAAGCCACAGTCCTC-3’. The agarose
gel-purified PCR amplicon was directly sequenced using both primers.
performed, according to a previously described procedure [24 (link)] with a minor modification. Briefly, tail DNA from the transgenic mouse was
digested with PstI and used for adaptor ligation. AmpliTaq360 master mix
(Life Technologies) was used for PCR. The oligonucleotides for a splinkerette adaptor are
5’-CGAAGAGTAACCGTTGCTAGGAGAGACCGTGGCTGAATGAGACTGGTGTCGACACTAGTGGtgca-3’ and
5’-CCACTAGTGTCGACACCAGTCTCTAATTTTTTTTTTCAAAAAAA-3’. Primers used for splinkerette-PCR are
as follows: 5’-CGAAGAGTAACCGTTGCTAGGAGAGACC-3’ (Splink1) and
5’-CATAATGCCAGGCGGGCCATTTACC-3’ (CMVIE-4R) for primary PCR amplification; and
5’-GTGGCTGAATGAGACTGGTGTCGAC-3’ (Splink2) and 5’-GGGCGTACTTGGCATATGATACACTTGATG-3’
(CMVIE-5R) for secondary PCR amplification.
The 3’ portion of the integrated transgene was PCR-amplified using CM32 Tg genomic DNA
and the primers 5’-TGCTCCTGGAGATGTTGGATG-3’ and 5’-TCAGTGAAGCCACAGTCCTC-3’. The agarose
gel-purified PCR amplicon was directly sequenced using both primers.
5
Validation of EGFR Exons 19 and 21 Alterations
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The presence of somatic alterations in EGFR exons 19 and 21 was validated by Sanger sequencing analysis in all cases displaying equivocal immunohistochemical stains, as recommended [29 (link), 30 (link)]. For this analysis, genomic DNA of each tumor and matched normal tissue was collected from the corresponding FFPE blocks and extracted using the DNeasy® Blood & Tissue Kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. Primers set to amplify all coding regions of exon 19 (chromosomal position: chr.7: 55,174,722–55,174,820) and exon 21 (chromosomal position: chr.7: 55,191,719–55,191,874) of the EGFR gene were employed as described [31 (link)]. PCR amplification of 60 ng of genomic DNA was performed using the AmpliTaq 360 master mix (Life Technologies, Grand Island, NY). PCR fragments were purified and sequenced on an ABI3730 capillary sequencer as described [32 (link)]. Sequences of the forward and reverse strands were analyzed using SeqScape (version 2.5, Applied Biosystems, Carlsbad, CA).
6
Targeted MED12 Exon 2 Sequencing
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