TITLE: DNA Concentration Protocol
AUTHORS: chyanne.rosenbaum

[DESCRIPTION]
This protocol is how the Eagle Fish Genetics Lab takes extracted DNA in a 96 well plate and concentrates it to five times the initial concentration. 

[GUIDELINES]
This creates a 5x concentration.


[STEPS]
SECTION: Section 1 - Prepare Your Plate
1. Supplies needed per concentration plate:
a.     Corresponding Initial DNA Extraction plate(s) 
b.     low profile non-skirted PCR tray
c.     p200 pipette (green) set to 25 µL
d.     p200 pipette (green) tips
e.     Nexttec sealing tape (clear plastic seal)


SECTION: Section 1 - Prepare Your Plate
2. Sign up for a thermocycler (1-2 hours)
SECTION: Section 1 - Prepare Your Plate
4. Using the p200, take 25 µL of initial DNA extract and dispense into the label low profile non-skirted PCR tray. 
a.     Use fresh tips for each sample to prevent contamination. 
b.     Pro tip – if you are not going to fill the whole tray, outline the working wells with sharpie.
SECTION: Section 1 - Prepare Your Plate
3. Label low profile non-skirted PCR tray: “Concentration”, date, tray #, project initials and your initials.
SECTION: Section 1 - Prepare Your Plate
5.      Seal the plate with clear plastic seal.
SECTION: Section 1 - Prepare Your Plate
6.      Balance and spin down at maximum speed (3700) for 5 minutes on centrifuge.
SECTION: Section 2 – Thermocycler
7. Turn on thermocycler by using the switch on the back right of the machine. 
SECTION: Section 2 – Thermocycler
10.  Place your plate into the machine.
a.     Keep the lid of the machine OPEN.
SECTION: Section 2 – Thermocycler
8.      Open the lid to the thermocycler.
SECTION: Section 2 – Thermocycler
9.      Remove clear plastic seal from your plate and discard the seal.
SECTION: Section 2 – Thermocycler
11.      Follow the string of commands below which corresponds to your thermocycler.

a.     PCT-100 (Machine #15)
i.    Select “Run” --> Press “Proceed” --> Select “<MAIN>” --> Press “Proceed” --> Select “70EVAPO” --> Press “Proceed” --> Select “NO” --> Press “Proceed” --> the machine will start.

b.     DNA Engine (Machine #16)
i.    Select “Run” à Press “Proceed” --> Select “<MAIN>” --> Press “Proceed” --> Select “EVAPO-85”--> Press “Proceed” --> Select “PLATE” --> Press “Proceed” --> next to the “VOLUME” enter “25” --> Press “Proceed” --> the machine will start.


SECTION: Section 2 – Thermocycler
12. Check after half an hour to see if all liquids have been evaporated, if not, continue to monitor.
a.     Avoid continuous heating of dry wells, do not over evaporate.
SECTION: Section 2 – Thermocycler
13.      Turn off thermocycler with switch in the back and the close lid. 
SECTION: Section 3 - Rehydrate
14. Supplies needed:
a.     Nuclease free water  
b.     Reservoir for the water
c.     p10 multichannel pipette (red) set to 5 µL
d.     p10 pipette tips (red)
e.     Heat seal 
SECTION: Section 3 - Rehydrate
16. Dispense 5 µL of nuclease free water into each of the evaporated working wells.
a.     Use a new tip for each well. 
b.     Do NOT use the hover techniques as some DNA may be aerosolized and can still contaminate the hovering tips.
SECTION: Section 3 - Rehydrate
15. Fill a reservoir a quarter of the way with nuclease free water.
SECTION: Section 3 - Rehydrate
17. Seal plate with heat seal
SECTION: Section 3 - Rehydrate
18. Vortex WELL, quickly spin down and let sit at room temperature for 5 min.