Genotyping of short tandem repeats (STRs)

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Bad quality of chemicals. Use new chemicals. Contamination of solutions. Prevent contamination (use gloves, work in biohazard box etc.) There are non-specific.
Genotyping of short tandem repeats (STRs) markers with 6 bp or higher length difference using PCR and high resolution agarose electrophoresis

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Laboratory of Molecular and Cellular Immunology, Institute of Molecular Genetics AS CR, v.v.i.,

Prague, Czech Republic. 2Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno, Czech Republic. Correspondence should be addressed to M.L. ([email protected]).

This protocol describes DNA typing of short tandem repeats (STRs), which differ in at least 6 bp using PCR and optimized high resolution electrophoresis. DNA preparation by NaOH method or by standard TRI reagent procedure takes about 4 hours. When DNA is prepared in master plates, a single person can test 192 samples in maximally 5 hours. It is suitable for quick test of interval-specific congenic strains, marker-assisted breeding of congenic mouse strains, test of presence of transgenes, knock-out or knock-in alleles in gene targeting technologies in breeding crosses, and for genotyping of intraspecific crosses, especially those derived from parents, which differ in limited percentage of their genomes.

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2 INTRODUCTION

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MATERIALS

REAGENTS



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23 ANTICIPATED RESULTS

24 ACKNOWLEDGMENTS

COMPETING FINANCIAL INTERESTS

25 REFERENCES

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27 TABLES

number of reactions* Forward primer 6.7 μM Reverse primer 6.7 μM 5x PCR buffer total Sterile distilled H2O Taq polymerase 1U/μl Total volume

1 6 (9) 9 (13) 12 (15) 14 (18) 18 (27) 24 (30) 24 (36) 37 (45) 36 (54) 48 (60) 0.34 3.06 4.4 5.1 6.1 9.2 10.2 12.2 15.3 18.4 20.4 0.34 3.06 4.4 5.1 6.1 9.2 10.2 12.2 15.3 18.4 20.4 4 36 52 60 72 108 120 144 180 216 240 4.9 44.3 64.0 73.8 88.6 132.8 147.6 177.1 221.4 265.7 295.2 0.4 3.6 5.2 6.0 7.2 10.8 12.0 14.4 18.0 21.6 24.0 10 90 130 150 180 270 300 360 450 540 600 units: μl

* number in brackets = theoretical number of reactions (without loss due to sticking of the liquid on the surface of tubes and pipettes)

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A

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B

2

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F

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G

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H

74

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Problem There is too much product at start of electrophoresis

There is no product

Possible reason Too much DNA in sample

Solution Dilute DNA sample before repeating of PCR

Bad conditions of reaction for certain primers

Optimize reaction conditions, usually different annealing temperature or Mg2+ concentration is necessary Use DNA from stock solution or isolate new DNA Optimize reaction conditions, usually usually different annealing temperature or different Mg2+ concentration is necessary Use new chemicals Prevent contamination (use gloves, work in biohazard box etc.) Optimize reaction conditions, usually different annealing temperature or Mg2+ concentration is needed

Bad quality of DNA sample Bad conditions of reaction for certain primers Bad quality of chemicals Contamination of solutions

There are non-specific products overlap with PCR product

Bad conditions of reaction for certain primers.

30 FIGURES