LABType® LABType®

Haik MuradyanHaik Muradyan

Key PointsKey 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 PointsKey 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 PointsKey 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 PurityMeasuring 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 PurityMeasuring DNA Concentration and Purity

CONCENTRATION:

DNA sample concentration range: 20-200 ng/µl.

DNA Concentration: A260 x Dilution Factor x 50 ng/µl

Example: 0.028 x 30 x 50 ng/µl

= 42 ng/µl

CONCENTRATION:

DNA sample concentration range: 20-200 ng/µl.

DNA Concentration: A260 x Dilution Factor x 50 ng/µl

Example: 0.028 x 30 x 50 ng/µl

= 42 ng/µl

Measuring DNA Concentration and PurityMeasuring DNA Concentration and Purity

PURITY:

DNA Purity = A260 / A280

Recommended range: 1.65 - 1.8

RNA contamination > 1.8

Protein contamination < 1.65

PURITY:

DNA Purity = A260 / A280

Recommended range: 1.65 - 1.8

RNA contamination > 1.8

Protein contamination < 1.65

Class I Gel PhotoClass I Gel Photo

2 major bands = Exon 2 and Exon 3

A locus: 571bp (Exon 2) and 348-349bp (Exon 3)

B locus: 579-594bp (Exon 2) and 333-338bp (Exon 3)

C locus: 582-585bp (Exon 2) and 370bp (Exon 3)

2 major bands = Exon 2 and Exon 3

A locus: 571bp (Exon 2) and 348-349bp (Exon 3)

B locus: 579-594bp (Exon 2) and 333-338bp (Exon 3)

C locus: 582-585bp (Exon 2) and 370bp (Exon 3)

A locus

B locus

C locus

Class I Gel PhotoClass 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

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 PhotoClass II Gel Photo

1 major band = Exon 2 only DRB1 locus: 266-288bp DRB3, 4, 5: 250bp DQB1 locus: 251-252bp Pseudogene bands are

typically not seen with Class II gels

1 major band = Exon 2 only DRB1 locus: 266-288bp DRB3, 4, 5: 250bp DQB1 locus: 251-252bp Pseudogene bands are

typically not seen with Class II gels

DRB1 locus

When to Consider Running a GelWhen 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

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

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