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.


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).

Department of Microbiology
School of Medicine
University of Washington
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