TITLE: MAD4HatTeR
AUTHORS: Andres Aranda-Diaz, eric.neubauervickers

[DESCRIPTION]
This protocol has been adapted from Paragon Genomics CleanPlex® NGS Panel

[STEPS]
SECTION: mPCR
1. In a PCR Workstation, prepare mPCR reaction mix

Keep Master Mix and thawed primers on ice

on ice Prepare a mPCR mix for your samples using the instructions below
Make fresh dilution of primers each time

Vortex reagents before using and spin down to collect liquids

Aliquot the mix into PCR tubes in a strip so that you can use a multichannel pipette and use smaller volumes. 


4 µL :
2 µL 
2 µL. You may dilute directly in the mPCR mix (e.g. for a 1:4 dilution, add 0.5 µL and1.5 µL)
  
 

Make at least 10% extra master mix to aliquot into wells. (e.g. for 10 samples, make 11 of the above):


Vortex, briefly spin and on ice aliquot 4 µL into PCR tubes/wells (single tubes, strips or plate)
SECTION: mPCR
2. Outside the PCR workstation, and ideally in a different area, add 6 µL to each labeled tube/well, independent of parasitemia.

Vortex and spin down before proceeding 

Cover with clear seal for transport to building 100

Take off clear seal and apply heat seal before putting in thermocycler; red line up.

10 µL 
SECTION: mPCR
3. Run PCR reaction on a thermocycler (ideally, in separate room)
Initial denaturation: 95 °C 10 min 
Denaturation: 98 °C 15 s with ramping 3 °C 
Annealing/Extension: 60 °C 5 min with ramping 2 °C 
Repeat Denaturation and Annealing/Extension for X total cycles (see below). 
 
Hold at 10 °C 
SECTION: mPCR
4. To prepare for following steps, after starting the thermocycler protocol you may want to:
Bring CleanMag Magnetic Beads and STOP buffer to Room temperature 
Bring index primers out of the freezer and thaw on ice 
Make mixes (step 6, step 9 and step 16) for digestion and indexing PCR
Make 70 % ethanol with nuclease-free water (35 mL ethanol + 15 mL water if using 50 mL conicals)
Make a plan for sample indexing. Write down what index you will use for each sample

If using TE at 37 C, move plate or tubes to  37 °C 
If using a 96-well pipettor, remove the tips corresponding to the empty wells (if any) on your plate. Save tips and put in used box for training purposes later.
SECTION: mPCR
5. Proceed to next section within 30 min 
SECTION: Post-mPCR bead purification
6. Spin the tubes/plate and add to each tube/well:
 2 µL 
 10 µL 

Vortex briefly and spin down

OPTIONAL:
You may make a mix with 1X TE and STOP buffer (10 volumes 1X TE and 2 volumes STOP buffer) and add 12 µL of that mixture to each tube. 


22 µL 
SECTION: Post-mPCR bead purification
7. Incubate PCR products with beads:
CleanMag Magnetic Beads should be at Room temperature before adding to the mixture.
If using beads other than CleanMag Magnetic Beads you have to adjust ratios

SECTION: Post-mPCR bead purification
7.1. Add 1.3 times the volume of the mixture in CleanMag Magnetic Beads (29 µL for 1 primer pool, 31 µL for 2 pools). Vortex vigorously to mix and incubate for 5 min at Room temperature

Do not vortex after this step and treat mixture carefully.

51 µL 

SECTION: Post-mPCR bead purification
7.2. Briefly spin down and place on magnetic stand for 3 min or until the beads are collected on the side of the tubes/wells and the liquid is clear. 
SECTION: Post-mPCR bead purification
7.3. Remove all the liquid: first with a P200 pipet (set to 180). Then briefly spin down and remove the liquid leftovers using a P20 pipet. 

PROTIP: Place the tubes in the spinner with the beads so that they are on the outside, further away from the center or axis of rotation. 
SECTION: Post-mPCR bead purification
8. Wash with 70% ethanol (use only freshly made 70% ethanol) 

SECTION: Post-mPCR bead purification
8.1. Add 180 µL 70 % volume ethanol 

To wash the beads, rotate the tubes/plate so that the beads migrate from one wall to the other. Incubate for 2 min or until all beads have migrated. You may need to CAREFULLY flick the tubes/wells

You can remove the liquid using the same tips you put it in with

SECTION: Post-mPCR bead purification
8.2. Remove the liquid with a P200 pipet and repeat wash   

SECTION: Post-mPCR bead purification
8.3. Spin down and remove all the remaining liquid using a P20 pipet (this is only on the second wash, not the first one)
SECTION: Post-mPCR bead purification
8.4. Leave tubes/wells open to dry Room temperature 
Generally, a 5 min dry time is enough, but it will depend on room temperature and humidity. The beads should look matte (right in figure), not shiny (left in figure). Under-drying (carrying ethanol) and over-drying (cracking) can lead to reduced yield

  

If you have only a few samples, you may want to keep an eye on each sample and close the tubes as they dry so that they all dry to the same extent.

SECTION: Post-mPCR bead purification
8.5. Add 10 µL 1X TE taken from the incubator. Close the tubes/wells and vortex vigorously to resuspend the beads. The magnetic beads will not affect the rest of the reactions. Quickly spin down. Return TE to the 37 C.

SECTION: Post-mPCR bead purification
8.6. This is a safe stopping point
If you want to stop here, store -20 °C 

SECTION: Digestion Reaction
9. Make Digestion Reaction Master Mix
If possible, perform this step in a PCR Workstation
on ice 
10 µL :
6 µL
2 µL 
2 µL 

* Buffer sometimes has a white precipitate after thawing. Make sure that it is completely dissolved before using

Make at least 10% extra master mix to aliquot into wells. (e.g. for 10 samples, make 11 of the above)

If you want it is possible to make both the digestion reaction master mix and the Indexing PCR Master Mix (step 16) at the same time. This is especially useful if the PCR workstation is not physically close to the post-pcr work area. 
SECTION: Digestion Reaction
10. Add 10 µL to each tube/well. Mix carefully by pipetting up and down at least 5 times or vortex vigorously for ~3 seconds. Avoid prolonged vortexing. Spin briefly to collect the liquid.

Note: Use a 12 channel pipette with 1-channel reservoir when doing a full plate; there is not enough mix for the a 96-well reservoir
SECTION: Digestion Reaction
11. Seal plate (do not use hot seal) and incubate at 37 °C for 10 min, ideally in a thermocycler. Do not incubate for shorter or longer. 

Prepare tips for remainder of experiment during at this time 

SECTION: Digestion Reaction
12. Immediately add 2 µL to each tube/well and mix by spinning briefly then vortexing. Spin again briefly to collect the liquid. 

22 µL 

SECTION: Digestion Reaction
13. Proceed to post-digestion bead purification immediately.
SECTION: Post-Digestion Purification
14. Perform a 1.3X bead purification     

SECTION: Post-Digestion Purification
15. Proceed immediately to indexing PCR reaction
SECTION: Indexing PCR reaction
16. Make indexing PCR reaction mix in a PCR Workstation
SECTION: Indexing PCR reaction
16.1. on ice Prepare an indexing PCR mix for your samples using the below

26 µL :
8 µL 
18 µL 

Make at least 10% extra master mix to aliquot into tubes/wells. (e.g. for 10 samples, make 11 of the above)

SECTION: Indexing PCR reaction
16.2. Add to the 10 µL left in the tubes/wells:
26 µL 
2 µL and 2 µL . OR 4 µL (Make sure to centrifuge indexing primers before adding)
Indexing primers MUST only contribute to one well per primer

40 µL 

Spin down before proceeding 

Heat seal

Note: If running a plate, use a reservoir to aliquot the mix.
SECTION: Indexing PCR reaction
17. Run PCR reaction on a thermocycler 
Initial denaturation: 95 °C 10 min 
Denaturation: 98 °C 15 s with ramping 3 °C 
Annealing/Extension: 60 °C 75 s with ramping 2 °C 
Repeat Denaturation and Annealing/Extension for 15 total cycles 
Hold at 10 °C 

SECTION: Pooling
18. Pool samples (make sure that indexes are compatible) by mixing them into a single tube. 
We recommend skipping using the following volumes if using 15 cycles in mPCR:
30 µL
20 µL
15 µL
6 µL
3 µL
SECTION: Gel purification
20. Run the eluted pool on a 2.5 % agarose gel
We recommend running for 1 h or more, at 140 V using TBE buffer

Use a ladder that allows identification of 200 and 400 bp bands

Cut the 400 bp band and extract using a gel purification kit
SECTION: Capillary electrophoresis and quantification
21. (to be filled)
SECTION: Pooling
19. Perform a 1X bead purification by adding magnetic beads to the combined sample.
Follow same steps as above but make sure that the volume of beads is the same than the pool volume
1X ratio should be 40 µL into 40 µL 

Elute into 40 µL