1. Dilute original genomic DNA prep as 1:50 in ddH2O.
   2 µl DNA prep
   98 µl ddH2O

2. use filter tips to assemble PCR reactions
   For single reaction:

   10x PCR buffer (no MgCl2)	2.0 µl
   50mM MgCl2*	0.6 µl
   dNTPs (10mM each)	0.4 µl
   Taq polymerase (1:10 diluted)	0.3 µl
   ddH2O	12.7 µl
   forward primer (5 pmole/µl)**	1.0 µl
   reverse primer (5 pmole/µl)	1.0 µl
   templates (1:50 diluted)	2.0 µl
   Total volume:	20.0µl

3. run PCR program conditions:

   step 1: initial denaturation:	94 oC	5 minutes
   step 2: denaturation:	94 oC	30 seconds
   step 3: annealing***:	55 oC	30 seconds
   step 4: extension:	72 oC	1 minute
   step 5: go to step 2		for another 34 cycles
   step 6: extension:	72 oC	3 minutes
   step 7: incubate at	10 oC	for ever

4. check 3 µl of PCR product on 1% 0.5X TBE gel.
   For 40 ml gel:
   40 ml 0.5X TBE
   0.4 g agarose
   8 µl of a 1:1000 dilution of Sybr Safe dye
   (Ethidium Bromide is not allowed in the MEAD lab)

5. clean up good PCR products using Qiagen PCR purification Kit****.
   
   1. add 5 volume of PB buffer (~85µl) to each sample, and mix well.
   2. transfer all solution to Qiaquick spin column.
   3. spin column in microcentrifuge at max speed for 1 minute.
   4. decant liquid in the collection tube.
   5. add 750 µl of PE buffer to each sample.
   6. spin column in microcentrifuge at max speed for 1 minute.
   7. decant the collection tube.
   8. spin column in microcentrifuge at max speed for 1 minute again to remove residual liquid.
   9. transfer column to a new 1.5 ml microcentrifuge tube (eppie) with proper labeling.
   10. add 150 µl of ddH2O to each column, and wait for 1 minute to elute PCR products.
   11. spin column in microcentrifuge at max speed for 1 minute.
   12. store eluant (in the microcentrifuge tube) at -20 oC until needed for running on automatic sequencer.

6. clean Qiaquick spin columns and collection tubes as following:
   
   1. rinse collection tube twice with ddH2O using a squirt bottle.
   2. rinse Qiaquick spin column three times with ddH2O using a sqirt bottle.
   3. assemble Qiaquick spin column in collection tube, then fill it up with ddH2O.
   4. spin column in microcentrifuge at max speed for 1 minute.
   5. decant the collection tube, then place Qiaquick column back in.
   6. spin column again in microcentrifuge at max speed for 1 minute.
   7. disassemble Qiaquick column and collection tube, and leave them air-dry in the wired basket in dish strainer.

Notes:
LocusLabeling on forward primerexpected proximate PCR product size (bp)annealing temperature (oC)MgCl2 concentration (mM)DM406-FAM240621DM58HEX280551.5DM946-FAM170581.5 reference:
http://www.mbg.cornell.edu/CM_Images/Uploads/MBG/aquadro_lab_unlocal.html

*: MgCl2 concentration may vary depending on each locus.

**: Each forward primer solution is a mixture of 80% non-labeled primer and 20% fluorescent labeled primer.

***: Annealing temperature may vary depending on the primer pair of each locus.

****: (cat. # 28106) Reuse the Qiaquick spin columns and collection tubes.