1. Perform PCR reactions in 50 µl using 2 µl of colony from plate C (primers are VNG26/27 or equivalents, enzyme ISC-Biolase or Promega-Taq).

2. Check PCR on gel, load according to standard loading scheme.

3. Log clones that did not yield product on appropriate sheet.

4. Purify and array PCR products on glass slides.

5. Select 8 clones from each plate for sequencing (usually one clone from each column with a good PCR product from gel analysis. If possible, include A1 and H12 to check orientation of plate).

Standard Mix:

Add 48 µl mix to 2µl cells in 96-well PCR plate (ISC, T-3049-1).

	Stock	Brand	Cat.no.	1x	100x	103x
Water		Sigma	W4502	40.8	4080	4202.4
10x Buffer	10x	ISC		5.0	500	515.0
MgCl2	50 mM	ISC		1.5	150	154.5
DNTP mix	20 mM	Pharmacia	27-2035-02	0.3	30	30.9
Primer-mix	30 µM	GibcoBRL		0.2	20	20.6
Biolase	5 U/µl	ISC	C-5002-500	0.2	20	20.6

Cycling:

Temperature	Time	Cycles
94°	5�	1
94° 58° 72°	30� 30� 4�	40
72°	10�	1
4°	Hold

When using an Eppendorf repeat pipetter, make enough for 103 reactions and dispense 50 µl per well.

This protocol is also used for the PE 9600. Run takes 4 hours on PE 9700 and more than 4.5 hours on PE 9600.

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GEL LOADING SCHEME

Check PCR products on 1% Agarose gel (2-3 µl PCR product in 15 µl total loading volume)

For gels with 42 tooth combs:

M A1 B1 A2 B2 � A11 B11 A12 B12 M

M C1 D1 C2 D2 � C11 D11 C12 D12 M

M E1 F1 E2 F2 � E11 F11 E12 F12 M

M G1 H1 G2 H2 � G11 H11 G12 H12 M

For gels with 50 tooth combs:

M A1 B1 A2 B2 � A11 B11 A12 B12 M E1 F1 E2 F2 � E11 F11 E12 F12

M C1 D1 C2 D2 � C11 D11 C12 D12 M G1 H1 G2 H2 � G11 H11 G12 H12

Marker is 1 kb ladder (GIBCO-BRL).

Run approximately 2 hours (bromophenol blue marker 2/3 down lane).