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

February 23 , 2009

The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for fragments smaller than 300bp. It also will remove the buffer and restriction enzymes from a restriction digest reaction.

1. Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
2. Transfer into the Zymo column (small clear guys)
3. Add 500uL of Ethanol and pipette up and down to mix
4. spin through, discard waste.
5. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
6. spin through, discard waste.
7. Add 200 uL of PE or Zymo Wash buffer
8. spin through, discard waste.
9. spin for 90 seconds, full speed to dry.
10. elute with water into a fresh Eppendorf tube

EcoRI/BamHI Digest of Wobble Products

For wobble products, you will digest the entire extension reaction-worth of DNA Since you started with 50uL of extension reaction, you should now have 50uL of eluted DNA in water.

• Set up the following reaction in a PCR tube:

 50uL eluted DNA
 5.7uL NEB Buffer 2
 1uL EcoRI
 1uL BamHI

• Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
• Incubate the reaction at 37 degrees on the thermocycler
• Proceed to another Zymo small fragment cleanup

The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for fragments smaller than 300bp. It also will remove the buffer and restriction enzymes from a restriction digest reaction.

1. Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
2. Transfer into the Zymo column (small clear guys)
3. Add 500uL of Ethanol and pipette up and down to mix
4. spin through, discard waste.
5. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
6. spin through, discard waste.
7. Add 200 uL of PE or Zymo Wash buffer
8. spin through, discard waste.
9. spin for 90 seconds, full speed to dry.
10. elute with water into a fresh Eppendorf tube

Ligation of EcoRI/BamHI digests

   * Set up the following reaction: 

6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL Vector digest
1uL Insert digest
0.5uL T4 DNA Ligase

   * Pound upside down on the bench to mix
   * Give it a quick spin to send it back to the bottom of the tube
   * Incubate on the benchtop for 30min
   * Put on ice and proceed to the transformation