PowerPlex® Fusion System

7.C. Direct Amplification of DNA From Storage Card Punches (continued)

Symptoms Causes and Comments

Peak height imbalance (continued) DNA was not accessible on nonlytic material. Small loci may amplify preferentially, with large loci dropping out. Pretreat nonFTA materials with PunchSolution™ Reagent to ensure that DNA is liberated from cellular proteins.

7.D. Direct Amplification of DNA From Swabs

The following information is specific to direct amplification of DNA from swabs after pretreatment using the SwabSolution™ Kit. For additional information about general amplification and detection, see Section 7.A.

Symptoms

Faint or absent allele peaks

Faint or absent peaks for the positive control reaction

Causes and Comments

Poor sample deposition. Shedding and collection of donor cells was variable. Increase cycle number.

Inactive SwabSolution™ Reagent. Thaw the SwabSolution™

Reagent completely in a 37°C water bath, and mix by gentle inversion. Store the SwabSolution™ Reagent at 2–10°C. Do not store reagents in the refrigerator door, where the temperature can fluctuate. Do not refreeze; avoid multiple freeze-thaw cycles, as this may reduce activity.

Active SwabSolution™ Reagent carried over into the amplification reaction. Ensure that the heat block is heating to

70°C (90°C if using a 2.2ml, Square-Well Deep Well Plate) and samples were incubated for the full 30 minutes. Incubation for shorter time periods may result in incomplete reagent inactivation. Do not use an incubator set at 70°C to incubate tubes or plates; heat transfer is inefficient and will result in poor performance. Use only a heat block to maintain efficient heat transfer. We have tested 60-minute incubation times and observed no difference in performance compared to a

30-minute incubation.

DNA was not accessible on nonlytic material. Pretreat nonFTA materials with SwabSolution™ Reagent to ensure that DNA is liberated from cellular proteins.

If the positive control reaction failed to amplify, check to make sure that the correct amount of 2800M Control DNA was added to the reaction. Due to the reduced cycle numbers used with swab extracts, it is necessary to increase the mass of

2800M Control DNA to obtain a profile. We recommend 10ng of 2800M Control DNA per 25μl amplification reaction. This mass of DNA should be reduced if the cycle number used is increased and decreased if the cycle number is increased.

Increase or decrease by twofold the mass of 2800M Control

DNA for every one-cycle decrease or increase, respectively.

Improper storage of the 2800M Control DNA.

Promega Corporation ·

2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com

Part# TMD039

Page 54

Printed in USA.

Revised 10/12

Symptoms

Extra peaks visible in one or all color channels

Peak height imbalance

Causes and Comments

Swab extract was contaminated. Assemble a reaction containing the swab extract prepared from a blank swab, or assemble a reaction where the SwabSolution™ Reagent is processed and incubated as a blank without a swab.

Artifacts of STR amplification. Amplification of swab extracts with high DNA concentrations can result in artifact peaks due to overamplification, resulting in saturated signal on the CE instrument. We recommend 2µl of swab extract per 25µl reaction. Using more than 2µl in a 25µl reaction or using 2µl with a smaller reaction volume may result in overamplification and signal saturation. If signal is saturated, repeat amplification with less swab extract or reduced cycle number.

Artifacts of STR amplification. Amplification of STRs can result in artifacts that appear as peaks one base smaller than the allele due to incomplete addition of the 3´ A residue.

• Be sure to perform the 20-minute extension step at 60°C after thermal cycling (Section 4.C)

• Use 2µl of swab extract in a 25µl PowerPlex ® Fusion reaction. A larger volume of swab extract may contain more than the recommended amount of DNA template, resulting in incomplete adenylation.

• Decrease cycle number.

• Increase the final extension time.

Excess DNA in the amplification reaction can result in locusto-locus imbalance within a dye channel such that the peak heights at the smaller loci are greater than those at the larger loci (ski-slope effect). Use less swab extract, or reduce cycle number.

Active SwabSolution™ Reagent carried over from swab extracts into the amplification reaction. Larger loci are most susceptible to reagent carryover and will drop out before the smaller loci. Ensure that the heat block is heating to 70°C

(90°C if using 2.2ml, Square-Well Deep Well Plates) and samples were incubated for the full 30 minutes. Incubation for shorter time periods may result in incomplete reagent inactivation. Do not use an incubator set at 70°C to incubate tubes or plates; heat transfer is inefficient and will result in poor performance. Use only a heat block to maintain efficient heat transfer.

Inactive SwabSolution™ Reagent. Thaw the SwabSolution™

Reagent completely in a 37°C water bath, and mix by gentle inversion. Store the SwabSolution™ Reagent at 2–10°C. Do not store reagents in the refrigerator door, where the temperature can fluctuate. Do not re-freeze; avoid multiple freeze-thaw cycles, as this may reduce activity.

DNA was not accessible on nonlytic material. Small loci may amplify preferentially, with large loci dropping out. Pretreat nonFTA materials with PunchSolution™ Reagent to ensure that DNA is liberated from cellular proteins.

Promega Corporation ·

2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com

Printed in USA.

Revised 10/12

Part# TMD039

Page 55