KSUD14-STS ASSAY FOR TAGGING LR21

New D14 primers.

Lr21L:

CGC TTT TAC CGA GAT TGG TC

Lr21R:

TCT GGT ATC TCA CGA AGC CTT

Lr21: 669 bp

lr21-Fielder: 757 bp

lr21-Wichita: 774 bp

Recipe (in 25 µl reaction).

Buffer (10X)	2.5 µl
2.5 mM dNTP	2.5 µl
25 mM MgCl	2.5 µl
DNA polymerase (Applied Biosystems AmpliTaq Gold; part number N8080243)	0.15 µl
Lr21L (1 pmol/ul)	1.0 µl
Lr21R (1 pmol/ul)	1.0 µl
DNA	50.0 ng
ddH2O	to 25.0 ml

PCR program.

1. 95°C for 8 minutes
2. 95°C for 30 seconds
3. 54°C for 1 minute
4. 72°C for 1 minute
5. repeat steps 2-4 34 times
6. 72°C for 7 minutes
7. 4°C to end

Gel electrophoresis. Separate the PCR products on a 2% agarose gel at 50V for 1 hour on a medium size gel. See figure at right for diagnostic fragment for Lr21.