SBB09Ntbk-Patrick Harrigan

February 28, 2009

For my Wobble product:

• Order the oligos, they don't need to be purified in any special way, smallest scale is ok
• Make 100uM stocks, this is the concentration used directly for the reaction
• Prepare the following reaction:

 29 uL water
 5 uL Expand 10x Buffer 2
 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
 5 uL Oligo 1 (100uM)
 5 uL Oligo 2 (100uM)
 0.75 uL Expand Polymerase 1

For my PCR Product:

The oligo concentrations in your stocks should be 100uM. You use them at 10uM in this protocol. So, you first need to make an oligo dilution of:

 9uL Water
 1uL 100uM oligo

You can throw away the remainder of the diluted oligo when you are done, but hold onto your stock tube!

Set up the following reaction in a PCR tube:

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA