Presentation is loading. Please wait.

Presentation is loading. Please wait.

LABType ® Haik Muradyan. Key Points Do I have to run a gel in conjunction with the LabType ® assay? Gel confirms successful amplification Ensures generation.

Similar presentations


Presentation on theme: "LABType ® Haik Muradyan. Key Points Do I have to run a gel in conjunction with the LabType ® assay? Gel confirms successful amplification Ensures generation."— Presentation transcript:

1 LABType ® Haik Muradyan

2 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

3 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

4 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

5 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

6 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

7 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

8 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

9 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

10 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

11 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

12


Download ppt "LABType ® Haik Muradyan. Key Points Do I have to run a gel in conjunction with the LabType ® assay? Gel confirms successful amplification Ensures generation."

Similar presentations


Ads by Google