By Irene Tiemann-Boege, Andrea Betancourt
This quantity info protocols for genetic, molecular, cytological, and bioinformatic equipment for settling on haplotypes. Haplotyping: equipment and Protocols courses readers via tools that at once sort haploid cells, difficult-to-resolve gene households, high-resolution, brief diversity haplotyping for specific loci, and long-range haplotyping for entire chromosomes or genomes. Written within the hugely profitable Methods in Molecular Biology series layout, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, easily reproducible laboratory protocols, and tips about troubleshooting and warding off identified pitfalls.
Authoritative and useful Haplotyping: equipment and Protocols, goals to supply researchers with an summary of experimental tools for haplotyping.
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Additional resources for Haplotyping: Methods and Protocols (Methods in Molecular Biology)
Prepare a reaction cocktail for eight 96-well PCR plates for the second round of allele-specific PCR, as below. Aliquot 9 μl to each well using an automatic pipette. Add 1 μl of diluted 1st round PCR amplifications to each well of the 2nd round PCR plate, using an eight channel pipette. Seal the plate and mix by centrifuging at 500 × g for 1 min. 8. 6, step 14). 2 % agarose gel containing ethidium bromide, to confirm the presence of amplification products. Pool the second round PCR products from each 96-well plate (900 μl) in a 2 ml tube.
07 crossovers/μl. Increasingly accurate estimates of crossover concentrations should then be determined by increasing the number of reactions with varying amounts of input DNA, as described below. , 24–96 wells), using the preliminary estimation of crossover concentration (c1) from step 17. 5) × (1/c1). 021 μl, to each of the 24 reactions. Prepare four PCR reaction cocktails (a-d), each for 25 reactions, with varying DNA template input amounts, as below. 5 °C/cycle) Annealing temperature Sequencing Arabidopsis Crossover Molecules 41 42 Kyuha Choi et al.
HyperLadder 1 kb (Bioline, H1-415106). 5× DNA loading buffer (Bioline, BIO37045). 4 Mapping Crossover Sites by Sanger Sequencing 1. Exonuclease (EXOI, New England Biolabs, M0293L 20 U/μl). Shrimp alkaline phosphatase (SAP, Takara, 2660A 1U/μl). 1 Cycle Sequencing Kit (Applied Biosystems). 5× BigDye sequencing buffer (Applied Biosystems). 10× SYBR green (Sigma-Aldrich, S9430). 4), 500 mM KCl, 15 mM MgCl2). GoTaq DNA Polymerase (Promega, M3001). Real time PCR thermal cycler (Bio-Rad CFX96).