Fingerprinting / fluorescent labeling

Part I. End labeling

**make sure to get right concentration when making mixtures**
***mix all tubes prior to using, then spin down, KEEP ON ICE***

Master mix:

Amount	Item	Company	P/N
3.2 ul	H2O
2.0 ul	Buffer #2 (10X concentration)	New England Biolabs	B7002S
1.0 ul	dATP (1 mM)	Amersham	27-2050-01
1.0 ul	ddGTP	Beckman DTCS Kit	608000
0.5 ul	polymerase	Beckman DTCS Kit	608000
0.1 ul	Hind III (10U at 100U/ul)	New England Biolabs	R0104L
0.2 ul	Hae III (10U at 18U/ml}	New England Biolabs	R0108M

Take above amount and multiply by number of samples, plus one

	1 plate	2 plates	4 plates	6 plates
H2O	336	672	1344	2016
NEB #2	210	420	840	1260
dATP	105	210	420	630
ddGTP	105	210	420	630
polymerase	52.5	105	210	315
Hind III	10.5	21	42	63
Hae III	21	42	84	126

Mix master mix by pipette, NOT inverting tube. KEEP ON ICE

Add 8 ul of mixture to each well. Then add 12 ul of DNA sample (be sure to mix DNA prior to taking samples). Add to well & mix slowly 5 times, then seal with Titer top (Diversified Biotech cat# T-TOPS-100)

Put into holder, counterbalance and centrifuge until reach 2000g, then stop. Make sure no air bubbles at bottom of wells.

Take out of centrifuge, put in thermocycler, 1 cycle {37^oC for 1 hour, 72^oC for 2 minutes, then 4^oC for infinity}.

Follow Beckman protocol in DTCS kit (only the EtOH precipitation part)
Make up solution for the appropriate number of rows

	For one row:	For one plate:
Water	16 ul	192 ul
0.5M EDTA	4 ul	48 ul
3.0M NaOAc	20 ul	240 ul
20mg/ml glycogen	10 ul	120 ul

Make master mix and mix by pipetting, NOT inverting. Then add 5 ul of mixture to each well. Add 60 ul of chilled 100% EtOH, put cover back on, vortex ~5 seconds at 5.5 speed. Let sit for at least an hour (I usually let sit overnight) at -20^oC

Centrifuge at 5000 rpm, for 15 minutes, at 4^oC

Gently dump out supernatant and blot, should see white precipitate.

IMPORTANT: Get as much EtOH out as possible. Beckman machines are very sensitive to salt & other ionic competitors

Add 200ul of 70% chilled EtOH, then centrifuge at 5000 rpm, for 3 minutes, at 4^oC. After remove from centrifuge, gently dump supernatant.

IMPORTANT: Again get as much EtOH out as possible

REPEAT 70% EtOH STEP, and then REPEAT 70% EtOH STEP AGAIN

After remove from centrifuge after the 3rd 70% EtOH wash step, gently dump supernatant

IMPORTANT: Again get as much EtOH out as possible. Then put into Speedvac and let dry 15-20 minutes (Needs to be completely dry, should not be able to see white pellet if it is completely dry)

CAN NOT STRESS THIS ENOUGH

Check formamide pH prior to using, if not 7.0 or above DO NOT USE!!! DO NOT assume Beckman supplied formamide is ok, sometimes it is NOT.

If pH is less than 7.0, you will see a decrease in signal intensity, getting worse as the pH drops in value. Can use Beckman formamide from DTCS kit or can make formamide (see protocol)

Use formamide (350ul/row, 700ul/2 rows, or 2500ul/plate) and 2 ul/row of 600 size standard (Beckman catalog # 608095)

Keep size standard on ice until used, mix and then spin down. Mix formamide and size standard together well with pipette. Do not refreeze any leftover formamide, need to dispose of properly as hazardous waste.

Ex: 16 samples (2 rows) = 400 ul (use a little extra),
440 ul formamide and 2 ul size standard

Resuspend pellet with 25 ul of formamide mixture per well, add one drop of mineral oil/well. Centrifuge until reach 2000 then stop. Check to be sure no air bubbles (especially between oil & sample)

Ideally run samples on that day, but can keep at 4^oC for several days. If going to be longer than a few days before running on machine, store at -20^oC. Generally better results if run within 3 days of labelling.