TITLE: Low-cost recombinase polymerase amplification (RPA)
AUTHORS: Smitha  Hegde

[DESCRIPTION]
This protocol describes the expression of enzymes and creation of a master mix for recombinase polymerase amplification (RPA) assays.

[STEPS]
SECTION: Dialysis and protein concentration
4. Dialyse the eluate with it's respective storage buffer. Follow the video in this link for instructions.
concentrate the elaute using a concentrator to required concentration (if your protein working concentration is 600ng/ul, atleast concentrate to 12-20 ug/ul)

 

SECTION: Column purification
3. Mix and load 6 ml of the resin (or any available Ni-NTA column) along with it's storage buffer and let the resin gravity settle. The millipore Ni-NTA agarose resin has 50% resin and the remaining 50% is loading buffer. So the column volume (CV) is 3ml in this case. Discard the flow through.
When the level of storage buffer reaches the top of the resin, equilibrate the column with following washes in sequence (i) 3 CV of MiliQ water (ii) 5 CV of 1X charge buffer. If your resin is precharged, skip this step (iii) 3 CV binding buffer.
Load the cell extract.
Wash the column with 10 CV of binding buffer.
Elute the bound protein through 10 CV of elute buffer, collect the eluate. Eluate can be stored in 4 °C until dialysed to storage buffer. The protein in eluate buffer is not stable for long in 4 °C or in the elution buffer, hence dialyse to storage buffer within a day or two of elution.
Strip the Ni from the column using the stripping buffer.
To re-use the resin same day , follow step 1 or Store column in 20% ethanol in4 °C



SECTION: Cell lysis
2. The following day, centrifuge the culture by spinning down to maximum g force and then resuspend in 10 mL of 1X ice cold binding buffer. Add 25-40KU of lyzozyme per g of cells to break cell wall. Note: Benzonase addition can help break E.coli DNA. Novagen protocol suggests to avoid it as it could be in final purified protein. Hence, a suggestion is to avoid adding as it could possibly interfere in RPA and Cas 12 assays. note: addition of protease inhibitor is optional according to many protocols, add according to the manufacture's instructions.
Sonicate on ice for few alternating cycles (30 cycles) of sonication and hold for 20 s each.
Centrifuge the lysate at maximum g force, collect the supernatant and 0.22 um filter syringe to get rid of traces of cell debris that could otherwise clog the column. If you have no 0.22um filter, repeat spinning down the supernatant again and use the supernatant for further steps.

SECTION: Culturing cells
1. Grow BL21 cells in LB media with appropriate antibiotics, overnight at37 °C with shaking (for long term, store the pellets in -80 °C). 
Inoculate 1 mL of the overnight culture to fresh 100 mL LB (whatever capacity required) with right concentration of antibiotics and continue growing at 37 °C whilst shaking.
At ~ OD600 of 0.5, induce the expression of the gene using100 mL IPTG and culture them at 15 °C overnight, with continuous aeration.


SECTION: RPA protocol
5. Add the below component, first incubate at 37 °C for 30 min to 60 min (always add enzymes at the end).
Measure the fluorescence for Eva green (or any other dye like SYBER, SYTO can be used for real-time monitoring of RPA) with excitation at 495 nm and emission at 520 nm