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E.coli DNA assay Kit

Specification

Intended Use

E.coli DNA assay Kit, which is produced by Hzymes, can be used as quantitative determination of E.coli cellular residual DNA in biological products (e.g. recombinant protein, antibodies, vaccines, etc) with qPCR detection principle on TaqMan fluorescent probes. The kit has the characteristics of high-speed and high

specificity. The detection method can be traced back to China pharmacopoeia method.

Reagents and Materials Provided

 

Storage and Stability

1. Period of validity: 24 months under specified storage conditions.

2. Transportation and storage: Store at-25~-15℃ for long term storage and use dry ice or ice bag to keep it cold during transportation.

Preparation of E.coli DNA Positive

Control standard curve samples

The concentration of E.coli DNA Positive Control is marked on tube wall label. Check the actual concentration before dilution. Dilute E.coli DNA Positive Control with DNA dilution buffer in kit by serial dilution method. The diluted concentration is as follows: 3000pg/μL, 300pg/μL,30pg/μL,3pg/μL,0.3pg/μL, 0.03pg/μL.

The specific operating steps are as follows:

  1. Take and thaw E.coli DNA Positive Control and DNA Dilution Buffer stored at -20℃. Vortex and mix slightly after completely meltedthen centrifuge rapidly for 2-5 seconds.  Take 6 low retention centrifuge tubes and mark them as ST0, ST1, ST2, ST3, ST4 and ST5. Prepare E.coli DNA standard samples according to the following table. Mix with vortex mixer then centrifuge rapidly for 2-5 seconds before starting the next serial dilution. Store the diluted standard sample at 2~8℃and it should be prepared when using.

The thawed but unused DNA Dilution Buffe can be temporarily stored at 2-8℃.

Preparation of spike-and-recovery

quality control samples (ERC)

Prepare the spike-and-recovery sample of E.coli DNA(ERC) as required. (It is suggested that the amount of added standard should be set to 2-30 times of the historical testing value of the sample with no spike). Take the preparation of ERC sample with 30pg E.coli DNA as an example. The specific operating steps are as follows:

  1. Add 100μL of testing sample into 1.5mL low retention centrifuge tube.  Add 10μL of ST3, vortex and mix well then mark the centrifuge tube as ERC testing sample.  ERC sample should be pre-processed together with the same batch of testing samples then ERC extracted solution sample can be prepared.
Preparation of negative control sample

Set negative control as specified by experiments, the specific operating steps are as follows: Add 100μL of matrix solution sample (or DNA Dilution Buffer) into 1.5mL low retention centrifuge tube and mark the tube as NCS. NCS should be pre-processed together with the same batch of testing samples then NCS extracted solution can be prepared.

qPCR reaction systems
  1. Calculate the required number of reaction wells according to the detected standard curve and the number of samples to be tested. Number of reaction wells= (standard curve samples of 5 concentration gradient+1 BLK + 1 NCS + testing sample + ERC testing sample) ×3. Calculate the required total amount of Compound of primers and probes and qPCR

Master Mix according to the number of reaction wells:

Compound of primers and probes = (number of reaction wells+2) ×5μL (including 2 wells of loss quantity) qPCR Master Mix = (number of reaction wells+2)×15μL (including 2 wells of loss quantity) Thaw reagents at room temperature. Prepare the reaction mix according to the calculated amount of Compound of primers and probes and qPCR Master Mix in step 2 above. Then vortex and mix slightly. 

Sample adding of qPCR reaction

  1. Take a 96-well PCR plate and add 20μL reaction mix into each well.  According to Table 4, add BLK, NCS, SAM, ERC, and then add 10μL of ST1, ST2, ST3, ST4 and ST5 DNA standard solution in due order. Three replicates were made for each sample.
  1. Seal the plate with adhesive membrane and centrifuge for qPCR reaction.