TITLE: Generating Ct cut-off values using gBlocks gene fragments
AUTHORS: Dilip Abraham, Nick Grassly, Catherine Troman, Jonathan Rigby

[DESCRIPTION]
The following protocol describes the resuspension, dilution, and qPCR of gBlocks gene fragments. gBlocks gene fragments are synthesised double stranded DNA oligos, which can be used for standardisation. 

In this protocol, the standard curves generated by the gBlocks in a triplex qPCR are used to determine a Ct cut-off value for the S.Typhi gene targets (ttr, tviB, staG) and the HF183 bacteroides rRNA gene.



[STEPS]
SECTION: g-blocks details
1. gBlocks gene fragments are synthesised double stranded DNA fragments which contain the sequence for the amplicon of interest, in this case for ttr, staG and tviB in S.Typhi, and HF183 bacteroides. 

 
 
SECTION: Resuspending and diluting the gblocks
2. gBlocks are supplied as a lyophilised pellet. Resuspend in TE buffer to achieve a stock of 10ng/uL. 
Information on the ng provided, OD260, and molecular weight are given on the spec sheet provided with the gblocks.

The following online tools can be used to assist in these calculations:
Integrated DNA Technologies - Resuspension Calculator (idtdna.com)
Oligo Analyzer (idtdna.com)
SECTION: Resuspending and diluting the gblocks
3. Once resuspended, check the concentration via Qubit or Nanodrop.
SECTION: Resuspending and diluting the gblocks
4. Create serial dilutions of your stock solution adding 2uL of stock into 18uL of nuclease free water. We recommend carrying out 12 dilutions to create a series of 12 concentrations. We recommend at least 10 replicates split over at least two days.
SECTION: qPCR and generating a standard curve
5. Prepare the triplex qPCR mastermix described below (or singleplex for HF183) 

 

 

SECTION: qPCR and generating a standard curve
6. Aliquot 20uL of master mix for each reaction in a 96-well plate. Add 5uL of gBlock dilution to each reaction. 

Ensure that although the reaction is designed as a triplex, you only put one target gBlock in each reaction.
SECTION: qPCR and generating a standard curve
7. Seal the plate carefully then spin down briefly to gather all reagents at the bottom of the wells and remove bubbles. 
SECTION: qPCR and generating a standard curve
8. Load the plate into the real-time PCR machine after setting it up appropriately and carry out cycling using the following conditions:

 

SECTION: Analysis - determining Ct cut-off
9. The limit of detection (LOD_95) is the genome copy number/uL and associated Ct value at which a qPCR amplification would be observed 95% of the time. This can be calculated from the results of the dilution series using PROBIT analysis. 
SECTION: Analysis - determining Ct cut-off
9.1. We have provided an Excel file to calculate the LOD_95 for you from your data (resource:   ). Make sure you allow macros to be run. You will also need to enable the Microsoft Solver add-in. Instructions for doing so are here.

Alternatively you can use the statistical programming language R to fit the PROBIT curve. Example code:


 



SECTION: Analysis - determining Ct cut-off
9.2. To calculate the GC/uL from the qPCR Ct values of actual samples you can use the equation: 

log GC/uL = (Ct – intercept) / slope

where slope and intercept are from the linear regression of the Ct value on log GC/uL generated from the standard curves (e.g. as given in the Excel spreadsheet or from the linear model (lm) fit in R).
SECTION: Resuspending and diluting the gblocks
4.1. For example, performing four replicates of 12 dilutions three times on three separate days. This would be two plates each day to include all targets.
SECTION: Resuspending and diluting the gblocks
3.1. If the concentration is not 10ng/ul, carry out your first dilution in step4 to make it 1ng/ul