LABType ® Haik Muradyan

Key Points Do I have to run a gel in conjunction with the LabType ® assay? Gel confirms successful amplification Ensures generation of optimal signals during hybridization Optional but recommended Do I have to run a gel in conjunction with the LabType ® assay? Gel confirms successful amplification Ensures generation of optimal signals during hybridization Optional but recommended

Key Points How do you run the gel? Procedure is done the same way as OLI SSP gel: Use 2.5g agarose in 100ml TBE buffer with 0.5µg/ml EB Use 30ml of the gel per gel box and 10ml of running buffer How do you run the gel? Procedure is done the same way as OLI SSP gel: Use 2.5g agarose in 100ml TBE buffer with 0.5µg/ml EB Use 30ml of the gel per gel box and 10ml of running buffer

Key Points The differences are: Use only 2-5µl of amplified DNA for electrophoresis Remove well combs during gel prep to allow sufficient spacing Run gel for approximately 10 minutes for complete band separation The differences are: Use only 2-5µl of amplified DNA for electrophoresis Remove well combs during gel prep to allow sufficient spacing Run gel for approximately 10 minutes for complete band separation

Measuring DNA Concentration and Purity DNA Concentration and Purity are critical when it comes to obtaining satisfactory results Use a spectrophotometer to measure properties DNA Concentration and Purity are critical when it comes to obtaining satisfactory results Use a spectrophotometer to measure properties

Measuring DNA Concentration and Purity CONCENTRATION: DNA sample concentration range: ng/µl. DNA Concentration: A 260 x Dilution Factor x 50 ng/µl Example: x 30 x 50 ng/µl = 42 ng/µl CONCENTRATION: DNA sample concentration range: ng/µl. DNA Concentration: A 260 x Dilution Factor x 50 ng/µl Example: x 30 x 50 ng/µl = 42 ng/µl

Measuring DNA Concentration and Purity PURITY: DNA Purity = A 260 / A 280 Recommended range: RNA contamination > 1.8 Protein contamination < 1.65 PURITY: DNA Purity = A 260 / A 280 Recommended range: RNA contamination > 1.8 Protein contamination < 1.65

Class I Gel Photo 2 major bands = Exon 2 and Exon 3 A locus: 571bp (Exon 2) and bp (Exon 3) B locus: bp (Exon 2) and bp (Exon 3) C locus: bp (Exon 2) and 370bp (Exon 3) A locus B locus C locus

Class I Gel Photo Exon 2 band of B locus is much lighter (fainter) than Exon 3 band – this is normal Multiple minor bands = due to the presence of pseudogenes which our primers recognize Presence of pseudogene bands do not interfere with typing results A locus B locus C locus

Class II Gel Photo 1 major band = Exon 2 only DRB1 locus: bp DRB3, 4, 5: 250bp DQB1 locus: bp Pseudogene bands are typically not seen with Class II gels DRB1 locus

When to Consider Running a Gel 1)After performing hybridization, check fluorescence data of beads and especially positive controls for Exon 2 and Exon 3. 2)If they are extremely low (considerably less than the threshold values), run a gel to confirm amplification was successful 3)If the sample in question looks like this, the amplification should be repeated 3 4)Now you may perform hybridization with the new amplified product DRB1 locus