Copy number standard curve. for absolute quantification by real-time PCR

Transcription

1 Copy number standard curve for absolute quantification by real-time PCR

2 Kit contents Positive control template for standard curve (RED) Template preparation buffer (YELLOW) RNase/DNase free water (WHITE) Reagents and equipment to be supplied by user Real-Time PCR Instrument Master Mix or Master Mix components This kit is designed to work well with all commercially available master mixes. However, we recommend the use of Primerdesign PrecisionPLUS or PrecisionFAST 2X qpcr Master Mix. Pipettors and tips Vortex and centrifuge Thin walled 0.5ml PCR reaction tubes Kit storage This kit is stable at room temperature but should be stored at -20ºC on arrival. Primerdesign does not recommend using the kit after the expiry date stated on the pack. Once the lyophilised components have been resuspended, unnecessary repeated freeze/thawing should be avoided. The kit components are stable for six months from the date of resuspension under these circumstances. The standard curve dilution series can be stored frozen for an extended period. If you see any degradation in this serial dilution a fresh standard curve can be prepared from the positive control. Dynamic range of standard curve This protocol provides for a standard curve with a dynamic range from 10 6 to 10 copies. Under ideal PCR conditions, 10 copies of the target will be detected. Primerdesign guarantees that a minimum of 100 copies will be detected. Copy number standard curve handbook 2

3 Bench-side protocol To minimise the risk of contamination with foreign DNA, we recommend that all pipetting be performed in a PCR clean environment. Ideally this would be a designated PCR cabinet. Filter tips are recommended for all pipetting steps. 1. Pulse-spin each tube in a centrifuge before opening. This will ensure lyophilised primer and probe mix is in the base of the tube and is not spilt upon opening the tube. 2. Resuspend the positive control template in template preparation buffer provided. To ensure complete resuspension, vortex each tube thoroughly. Component Post-PCR heat sealed foil Positive control template (RED) * Volume 500 μl * This component contains high copy number template and is a VERY significant contamination risk. It must be opened and handled in a separate laboratory environment, away from the other components. 3. Preparation of standard curve dilution series 1) Pipette 90μl of template preparation buffer into 5 tubes and label ) Pipette 10μl of positive control template (RED) into tube 2. 3) Vortex thoroughly. 4) Change pipette tip and pipette 10μl from tube 2 into tube 3. 5) Vortex thoroughly. Repeat steps 4 and 5 to complete the dilution series. Standard curve Copy number Tube 1 positive control (RED) 2 x10^5 per µl Tube 2 2 x 10^4 per µl Tube 3 2 x 10^3 per µl Tube 4 2 x 10^2 per µl Tube 5 20 per µl Tube 6 2 per µl Copy number standard curve handbook 3

4 Preparation of standard curve plate Each well of the standard curve should contain the reagents below. Make up a reaction mix containing master mix and primer/probe mix. This reaction mix needs to be enough for all wells. Pipette this onto your plate according to your layout. Finally pipette the standard curve template dilutions, starting with the lowest copy number first. Component 1 reaction PrecisionPLUS or PrecisionFAST Master Mix 10µl Primer/probe mix 1µl RNase/DNase free water (WHITE) 4µl Standard curve tube 1-6 5µl Final volume 20µl Copy number standard curve handbook 4

5 qpcr amplification protocol Please select the correct cycling protocol for the custom qpcr assay that you are using. Amplification conditions using PrecisionPLUS 2X qpcr Master mix For use with primer/probe (TaqMan ) gene detection kits Denaturation 10s 95ºC DATA COLLECTION* 60s 60ºC *Fluorogenic data should be collected during this step through the FAM channel. For use with SYBR Green gene detection kits Denaturation 10s 95ºC DATA COLLECTION* 60s 60ºC Melt Curve** *Fluorogenic data should be collected during this step through the SYBR Green channel. **A post PCR run melt curve can be used to prove the specificity of the primers. See the manufacturer s instructions for your hardware platform Copy number standard curve handbook 5

6 Amplification conditions using PrecisionFAST 2X qpcr Master Mix For use with primer/probe (TaqMan ) gene detection kits Denaturation 5s 95ºC DATA COLLECTION* 20s 60ºC *Fluorogenic data should be collected during this step through the FAM channel. For use with SYBR Green gene detection kits Denaturation 5s 95ºC DATA COLLECTION* 20s 60ºC Melt Curve** *Fluorogenic data should be collected during this step through the SYBR Green channel. **A post PCR run melt curve can be used to prove the specificity of the primers. See the manufacturer s instructions for your hardware platform Copy number standard curve handbook 6

7 Interpretation of results Amplification plots of standard curves Standard Curve The slope of the curve should be approximately indicating a priming efficiency of close to 100%. Copy number standard curve handbook 7