MycAway™ Mycoplasma qPCR Detection Kit -40618ES

Instructions

1.  Preparations before experiment

1） Prepare the required reagents and materials which will be used in the experiment.

2）Confirm the suitability of the qPCR instrument

This kit can be used on the types of qPCR instrument as below:

1. Bio-Rad: CFX96
2.     Thermo Scientific: 7500 Real-Time PCR System；QuantStudio™5；
3.  Experiment method

1） DNA Extraction

We recommend to use ‘ Magnetic Residual DNA Sample Preparation Kit’  (Cat#18461ES for manual extraction and Cat#18462ES for auto extraction) for the DNA extraction, you can visit  ‘ http:/www.yeasenbiotech.com ’  for the

detailed information and the purchasing.

The kit (Cat#40618ES) contains an internal control (IC). If add the IC to the samples prior to DNA extraction, it can verify the complete process (included DNA extraction and qPCR reaction). If add the IC to the qPCR master mix

directly, the IC will acts as a qPCR control only.

2）qPCR Mix preparation

1.     According to the sample amount which included Positive control (PCS), No template control (NTC), Negative   control solution (NCS) and Testsample (TS) to calculate the number of reactions. Prepare 2 reactions in parallel for

each sample in generally.

* PCS：Positive control solution；NTC：No template control；NCS：Negative control solution；TS：Test sample. There is no need to perform

the sample extraction for PCS and NTC, but NCS and TS are needed.

Reaction wells（M1）=（1×NCS + N×TS） ×2

Reaction wells（M2）=（1×PCS + 1×NTC） ×2

Reaction wells（M3）=（1×PCS + 1×NTC+ N×TS） ×2

1.     Pre-thaw the required amount reagents on ice according to the experiment design and Tables below.
2.     Calculate the amount of qPCR Mix according to the Number of reactions. Please note, ifthe kit will be used forGMP activities such as product release, we recommended used Table 1 and 2 for the preparation; If the kit will just  used for research and there is no need to add IC before the extraction after the evaluation, then follow Table 3 for

the preparation. Please note that not all M1, M2 and M3 are needed to be prepare.

Component	Volume（1×40μL Reactions）	Volume（M1×40μL）
4×  MyqPCR Reaction Buffer	10 μL	(M1+2)  ×10  μL
MyPrimer & Probe MIX	1 μL	(M1+2)  ×1  μL
ROX	0.8  μL /0  μL**	(M1+2)  ×0.8  μL /0  μL
Purified water	Up to 20  μL	Up to (M1+2)  ×20  μL
Total	20 μL	(M1+2)  ×20  μL

Table 1 qPCR Mix system for M1

 

*The configuration system in Table 1 is based on the premise that IC is added before extraction for both NCS and TS, so it is not necessary

to add IC when qPCR Mix preparation. IC adding method before extraction: Firstly, dilute IC by 20 times with DNA diluent, and add 1μL

diluted IC into each 100μL test sample for the further extraction.

**This kit does not contain ROX Reference Dye. If ROX reference dye is needed for the Real Time PCR amplifiers that you are currently using, 50×ROX Reference Dye (Cat#10200ES) is recommended for use. In this case, the added volume is 0.8μL, as shown in Table 1. If using other

brands of ROX products, please refer to their instructions for ROX addition. If no ROX reference dye is required, the added volume is 0  μL.

 

Component	Volume（1×40μL Reactions）	Volume（M2×40μL）
4×  MyqPCR Reaction Buffer	10 μL	(M2+2)  ×10  μL
MyPrimer & Probe MIX	1 μL	(M2+2)  ×1  μL
Internal Control（IC）	1 μL*	(M2+2)  ×1  μL /0  μL
ROX	0.8  μL /0  μL**	(M2+2)  ×0.8  μL /0  μL
Purified water	Up to 20  μL	Up to (M2+2)  ×20  μL
Total	20 μL	(M2+2)  ×20  μL

Table 2 qPCR Mix system for M2

*The configuration system in Table 2 is based on the premise that IC is not added in PCS and NTC before extraction, so IC needs to be added during qPCR Mix preparation. IC adding method after extraction: Dilute IC by 100 times with DNA diluent and add 1μL diluted IC into

each qPCR Mix system.

**This kit does not contain ROX Reference Dye. If ROX reference dye is needed for the Real Time PCR amplifiers that you are currently using, 50×ROX Reference Dye (Cat#10200ES) is recommended for use. In this case, the added volume is 0.8μL, as shown in Table 1. If using other

brands of ROX products, please refer to their instructions for ROX addition. If no ROX reference dye is required, the added volume is 0  μL.

 

Component	Volume（1×40μL Reactions）	Volume（M3×40μL）
4×  MyqPCR Reaction Buffer	10 μL	(M3+2)  ×10  μL
MyPrimer & Probe MIX	1 μL	(M3+2)  ×1  μL
Internal Control（IC）	1 μL*	(M3+2)  ×1  μL /0  μL
ROX	0.8  μL /0  μL**	(M3+2)  ×0.8  μL /0  μL
Purified water	Up to 20  μL	Up to (M3+2)  ×20  μL
Total	20 μL	(M3+2)  ×20  μL

Table 3 qPCR Mix system for M3

*The configuration system in Table 3 is based on the premise that IC is not added to samples before extraction, so IC needs to be added    during qPCR Mix preparation. IC adding method after extraction: Dilute IC by 100 times with DNA diluent and add 1μL into each qPCR Mix

system.

**This kit does not contain ROX Reference Dye. If ROX reference dye is needed for the Real Time PCR amplifiers that you are currently using, 50×ROX Reference Dye (Cat#10200ES) is recommended for use. In this case, the added volume is 0.8μL, as shown in Table 1. If using other

brands of ROX products, please refer to their instructions for ROX addition. If no ROX reference dye is required, the added volume is 0  μL.

3）Templates adding

1.     Mix the qPCR Mix with sufficient shaking, centrifuge at low speed and collect the residualliquid from the cap

to the bottom of the tube.

1.     Add 20  μL qPCR Mix to each reaction tube/well. Please note adding the corresponding qPCR Mix into each

sample tubes and avoid adding errors.

1.     AddTemplates to the tube/wells which contained the qPCR Mix. See Table 4 for the templates adding.

Test Samples	In each tube or well …
TS	20  μL qPCR Mix+20  μL Samples after extraction
NTC	20  μL qPCR Mix+20  μL DNA Dilution buffer
NCS*	20  μL qPCR Mix+20  μL Negative sample after extraction*
PCS	20  μL qPCR Mix +20  μL Positive control

Table 4 Templates adding

*We recommend to use DNA Dilution buffer (40618-E) as the template of NCS for the DNA extraction.

**The total reaction volume in each tube/well is 40  μL.

***Cover the tube lid or the plate film. To avoid affecting the fluorescence signal reading, please take care not to mark the tube lid or film

or even rub the film repeatedly with a scraper.

****Centrifuge the reaction tube or plate briefly at low speed after templates adding. After sufficient shaking and mixing, repeat centrifuge

at low speed to collect the liquid from the lid or wall to the bottom. Avoid bubbles when operation. The baseline will be impacted if the mix

is not mixed well, so this step is very important to a good experiment result.

4）qPCR programs setting

1. Program file Settings

Example (7500 Real-Time PCR System instrument and Real-Time PCR Software v2.4):

Instrument type：7500 (96 Wells)

Experiment type：Quantitation-Standard Curve；

Chemistry：Taqman® Reagents

Ramp Speed：Standard (~2 hours to complete a run)

1.     Target channel Settings

In "Define Targets and Samples" of "Plate Setup", create a Target 1 channel (FAM), select FAM as the reporting fluorescence group and MGB or none as the quenching fluorescence group. Create a Target 2 channel (VIC), select the reporting fluorescence group as VIC and the quenching fluorescence group as none. In "Assign Targets and Samples" of "Plate Setup", if no additional ROX dye is added, select "none"; If an additional ROX is added, select

ROX.

1.     Standard amplificationprogram Settings

S/N	Reaction Stage	Temperature	Time	Cycle(s)
1	Initial denaturation	95℃	5 min	1
2	Denaturation	95℃	15 sec	45
3	Annealing/Extension (fluorescence signal collection)	62℃	30 sec

Table 5 Standard amplification program Settings

1.     Baseline and threshold setting:

Principle of baseline adjustment: use the automatic baseline generally. If need to adjust in manual, choose the cycle before the exponential growth period as the start cycle, and avoid the fluctuation zone of initial fluorescence collection. Choose the cycle which is 1-2 cycles before the Ct of the earliest exponential amplification sample as the

end point.

Principle of threshold adjustment: use the automatic threshold generally. If need to adjust in manual, the threshold should be set higher than the Negative sample or the baseline noise, it’s generally set the threshold in the late

stage of the exponential amplification, relative independent and suitable threshold is needed for each tunnel.

5） Result Analysis

1. Resultjudgement for PCS, NTC and NCS:

If IC added: the specification of each control samples should be satisfied in Table 6:

Control sample	FAM Signal	VIC Signal
PCS	Ct < 40, and has obvious amplification curve	Ct < 40 and has obvious amplification curve
NTC	Ct  ≥ 40 or no obvious amplification curve	Ct < 40 and has obvious amplification curve
NCS	Ct  ≥ 40 or no obvious amplification curve	Ct < 40 and has obvious amplification curve

Table 6 Result judgement of PCS, NTC and NCS

If IC not added: each quality control sample shall meet the specification of FAM signal column in Table 6, and no

need to analyze VIC channel.

1. Resultjudgement for TS

Prerequisite: It is necessary to determine whether PCS, NTC and NCS passed the specification in Table 6 before TS results analysis. If passed, then can proceed to the next step. If not passed, the TS results may not be reliable, and

the reason needs to be investigated.

If IC added: find the corresponding results judgement according to the result information of FAM and VIC in Table 7:

FAM Signal	VIC Signal	Result judgement
Ct<40 and has obvious amplification curve	Ct<40 and has obvious amplification curve	Positive
	Ct≥40 or no obvious amplification curve	An inhibition is existed, the experiment need to be repeated
Ct≥40 or no obvious amplification curve	Ct<40 and has obvious amplification curve	Negative
	Ct≥40 or no obvious amplification curve	An inhibition is existed, the experiment need to be repeated

Table 7: Result judgement of TS (IC added)

*If there is inhibition for VIC signal,treatment is needed to eliminate the inhibitors or repeat the test.

If IC not added: find the corresponding results judgement according to the result information of FAM in Table 8 , and it is no need to analyze the VIC signal.

FAM Signal	Result judgement
Ct<40 and has obvious amplification curve	Positive
Ct≥40 or no obvious amplification curve	Negative

Table 8: Result judgement of TS (IC not added)

Notes

1.  Please read thismanual carefully before usingthis kit. The experiment should be conducted in a standardized manner, including sample handling, preparation of reaction system and sample addition.

2.  Keep operations ofsample adding and reagents preparing on ice if possible.
3. Vortex and mix well for each reagents before use.

4.  Please operatewith lab coats and disposable gloves，for your safety.
5. This product is for research use only.

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