Working Procedures

Q: Can I re-use the Eluent from the affigene® DNA extraction kit once it has been warmed to 70 degrees?

A: It should be OK to re-use the Eluent once it has been in 70 degrees but to be on the safe side, aliquot the exact amount needed before extraction.

Q: Can I use the results of the controls (non-template control and positive control) from a previous affigene® tracer run?

A: No, each run is unique and the positive control must be included in each run to set the threshold for the target signals. The non-template control must be extracted along with the clinical samples to be sure that no contamination has occurred.

Q: Do I need to run the controls (non-template control and positive control) each time, when running an affigene® tracer assay?

A: Yes, it is important to include both controls for each unique run. The positive control is used to set the threshold for the target signals and for checking the validity of the run. The non-template control is always needed to be sure that there is no contamination during the procedure.

Q: Can I use sealing film instead of the clear strip caps to my 96-well plate in the MX3000P?

A: Yes, you may use sealing film, but not all sealing films work. One that works is ClearSeal Strong optically clear sealing film (AB-0685) (ABgene). A sealing film should be used in combination with a pad to avoid evaporation.However the clear strip caps is recommended to use since they close tighter to the tubes and cause less evaporation.

Q: During extraction I added wash buffer 2 before wash buffer 1, what will happen?

A: You will not obtain any DNA. You are shocking the system by adding too low a salt concentration, and you will elute DNA and IC. You have to do the extraction again.

Q: I started a run with the default thermal profile for 7 cycles before I realized my mistake. Is it possible to start all over again with the correct thermal profile and the same PCR reactions?

A: No, it is not possible. The default setting is a three-step thermal profile and the correct one is a two-step profile. Also, you will already have a PCR product in the reaction tubes at start, the background signal will be high and the software will not set the individual baselines for each sample in an optimal way.

Q: Do I need to run all three controls (non-template control, calibrator Low and calibrator High) each time, when running an affigene® trender assay?

A: Yes, it is important to include both calibrators High and Low in order to calculate the standard curve correctly for each unique run. The non-template control is always needed to be sure that there is no contamination during the procedure.

Q: Can I use the results of the controls (non-template control, calibrator Low and calibrator High) from a previous affigene® trender run?

A: No, each run is unique and the two calibrators create the standard curve for each run.The non-template control must be extracted along with the clinical samples to be sure that no contamination has occurred.

Q: How do I know that the PCR reaction has been successful?

A: The assay is provided with a built-in security incorporating an internal control all the way from sample preparation for the monitoring of inhibition and sample recovery minimizing the risk for false negative results.

Q: How many times can the Master Mix be frozen and thawn?

A: The Master Mix must not be refrozen and rethawn. Once a tube of Master Mix has been thawn it must be used and any excess reagent must be discarded. Always discard single-use reagents after opening to avoid cross contamination.

Q: Do I need to work on ice with the Master Mix?

A: No, but you should take the Master Mix tube from the freezer just before use.During specimen preparation store the working Master Mix in 2-8°C, but not for more than four hours.

Q: There is a white precipitate in the working lysis buffer, what should I do?

A: A white precipitate may form in the working lysis buffer upon storage. If this happens dissolve the precipitate by incubating the bottle at 70°C before use.

Q: For how long is the working lysis buffer containing internal control (IC) stable?

A: Stability for working lysis buffer containing internal control is four hours in room temperature.

Q: How can I avoid rough baselines that sometimes occur in the beginning of a run?

A: Add the template close to the surface of the working Master Mix to avoid sample on the tube wall and/ or use a micro centrifuge to collect the sample in the bottom of the tube.

Q: How long does it take to perform an assay?

A: The results can be achieved within one day.
Specimen preparation:
- 15 clinical samples takes 1,5-2 h using centrifugation/vaccum or centrifugation protocol
- 45 clinical samples takes 2,5-3,5 h using centrifugation/vaccum or centrifugation protocol PCR: 2 h.

Q: How can I avoid cross-contamination between runs?

A: By following the instructions for work flow in the User Manual.In addition we utilize UDG sterilization and single-use reagents tominimize the risk for cross-contamination and thereby false positiveresults. Always discard an opened tube after use.