Sample Preparation Protocols

TwistDx RPA Kit Protocol*

For each sample, prepare the rehydration solution as follows:
• Primer A (10μM) 2.4 μl
• Primer B (10μM) 2.4 μl
• Rehydration Buffer 29.5 μl
• Template and dH2O 13.2 μl
• (Total Volume 47.5 μl)
Vortex and spin briefly.
1. The components of the rehydration solution can be combined in a master-mix for the number of samples required. In some circumstances, for example when performing a primer screen, a number of different rehydration solutions have to be made (here according to the number of primer pairs being tested). In that case components common to all reactions (e.g. template, rehydration buffer, water) should be prepared as a master-mix, distributed in a corresponding volume into fresh tubes, and be combined with the required volume of the different primer pairs. The different rehydration solutions are then used as normal according to the protocol.
NOTE: Primers and probes should be added simultaneously to pellets to avoid any bias in recombination filament formation.www.twistdx.co.uk TwistAmp® Basic kit
2. For each sample, transfer 47.5 μl of the rehydration solution to the reaction pellet. Mix by pipetting up and down until the entire pellet has been resuspended.
3. For each sample, add 2.5 μl 280 mM magnesium acetate and mix well. One way to do this simultaneously for many samples is to place the magnesium acetate into the lid of the reaction tubes (strip of 8) cap the tubes carefully and spin the magnesium acetate into the rehydrated material to initiate the reactions. Invert vigorously 8-10 times to mix and spin down once again.
4. Insert the tubes into a suitable incubator block (optimum 37-39°C) and incubate for 4 minutes.
5. After 4 minutes, take the samples out of the incubator, invert vigorously 8-10 times to mix, spin down and return the samples to the incubator block. (VARIATION IN THE EXACT TIME OF SAMPLE AGITATION CAN SOMETIMES IMPROVE PRODUCT FORMATION).
6. Continue the incubation/detection for a total incubation time of 20-40 minutes. If a timecourse of TwistAmp® Basic reaction is being taken the incubation time has to be adjusted as required. At the end of the incubation proceed to “Monitoring TwistAmp® Basic amplification reactions”.

Optimized RPA Protocol

The RPA Kit from TwistDx used in our experiments was designed to rapidly amplify short (~200bp) sequences. For our system we required longer amplicons and attempts were made to adjust the parameters to promote the amplification of longer sequences. Overall the adjustments were intended to reduce the reaction rate and prolong the lifetime of the reaction before exhaustion of resources. The following changes were made:
• Addition of 1mM ATP
• Addition of 0.1mM dNTP’s
• 2.15 μl of 280mM Mg-acetate instead of 2.5 μl
• Vigorous shaking after 6 minutes after initiation rather than 4 minutes

KAPA Blood PCR Kit Protocol**

**Taken from the KAPA Blood PCR Kit Manual

Whole human EDTA blood may be added to a final volume of 1 - 20% in the PCR reaction (i.e. 0.5 - 10.0 µl in a 50 µl reaction). Reaction volumes ranging from 10 to 50 µl may be used. Thorough mixing of the blood and other reaction components prior to thermal cycling is important.

A typical reaction is set up by mixing the components in the order listed in the table below. Once pipetting has been completed, shake or spin tubes briefly to collect all components in the bottom of the tube. Vortex to mix but do not spin again before reactions are placed in the thermocycler.



A typical 3-step cycling profile for Whole Blood PCR with KAPA Blood PCR Mix A or B is given in the table below.



Hybrid Blood & isoPCR Protocol

The sample preparation stage in our final system primarily consists of the RPA kit components; the optimized RPA protocol was used as the initial mixture. 25% v/v substitution of the total volume of the RPA reaction mixture was substituted with the Blood PCR Kit Mix A – a mixture containing all necessary components of the blood-compatible PCR except for template and primers.

Template may be either whole cells, as performed in typical colony PCR, or a purified DNA extract (e.g. via plasmid miniprep or genomic DNA extraction).