1. a) Get full tub and bucket of ice, b) get isotope out if in -20°C
2. Get dGTP or dITP master mix buffer and reaction buffer out and thaw on ice
3. Thaw primers on ice, they need to be 2 pmol/l
4. Label 4 PCR tubes per sample in the following order and color code:
   

   Base Letter	Marker Color	Description
   A	black	all colors
   C	red	crimson
   G	green
   T	blue	teal

   
   
5. Label 4 colored tubes for termination mixes and _X_ colored tubes for sample reaction mixes
6. Get dNTPs out to thaw
7. Do termination mix calc. using kit quick card as preparing for (n + 1) samples
8. Do reaction mix calc. (normally 1 l of mini prep is used, so adjust H₂O)
9. Transfer 2.5 l termination mix to each appropriate tube (left to right)
10. Transfer 4.5 l reaction mix to each appropriate tube (front to back) and mix sol'n with pipet tip
    
    Tub diagram:

    	T	T	T	T	T
    Hot	G	G	G	G	G
    bases	C	C	C	C	C
    	A	A	A	A	A
    Samples:	1	2	3	4	5

    
    
11. If using "Bonnie" don't need to overlay with oil
12. Thaw stop sol'n at end of cycles and add 4 l to each tube using single tip
13. Spin pulse all samples
14. Store at -20°C in refrig freezer

Notes:

• Make sure all "hot" tips and bullets get put into the radioactive solid waste
• In our experience, the reaction will not work if the buffers are switched
• It is very easy to get confused, allow yourself plenty of time to set up and mix the reactions; they can be done the day before and froze