Template:SBB-SOEing

Full SOEing Run

Here is the complete SOEing reaction guide.

Normal PCR

Cloning by PCR
This is the basic PCR method to amplify your DNA from a plasmid or genomic DNA sample using the Expand polymerase. You also use this protocol for an EIPCR reaction.

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

The Expand temperature programs are a little complicated, so I won’t write them out here. They all involve 2 distinct cycling phases—the first 10 cycles have shorter extensions than the latter 20 cycles. You choose which one you want based on the length of your predicted product. If it is under 2kb, use 2K55. If it is between 2kb and 4kb, use 4K55. If it is over 4kb, use 8K55. In each case, start with the "55" version of the program. If you get no product or poor yield of product, repeat the same PCR reaction as two different variants, both with the “45” program. The first reaction will be the same composition as the first. For the second one, don’t add 3.3 uL of the water and instead use the volume for 3.3 uL of DMSO (dimethylsulfoxide). If these PCRs fail as well, well you'd either redesign, use a different template source, or give up.

Run Gel then Zymo Gel Purification

• All spins until the drying step are 15 second full speed spins.

1. cut out bands minimizing extra gel matter.
2. put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
3. heat at 55, shake and/or vortex until the gel has dissolved.
4. If the DNA is <300bp add 250uL of isopropanol
5. transfer into the Zymo column inside a collection tube (small clear guys)
6. spin through, discard waste.
7. Add 200 uL of PE buffer (which is basically 70% ethanol)
8. spin through, discard waste.
9. Add 200 uL of PE buffer
10. spin through, discard waste.
11. spin for 90 seconds, full speed to dry.
12. elute with 8.5 uL of water into a fresh Eppendorf tube

SOEing PCR

• Set up PCR reactions according to your construction file as normal 33uL reactions as described in Cloning by PCR
• For each PCR, load 8uL of PCR product premixed with 1uL of loading buffer in a single well of a 1% agarose gel
• Cut out the bands, put them into a single 1.5mL microcentrifuge tube
• Add 650uL of ADB Buffer
• Proceed with the Zymo Gel Purification procedure
• Elute the DNA in 50uL of water
• Set up your second round of PCR as a normal 33uL reaction using the eluted mixture of fragments as template

Run Gel then Zymo Gel Purification

• All spins until the drying step are 15 second full speed spins.

1. cut out bands minimizing extra gel matter.
2. put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
3. heat at 55, shake and/or vortex until the gel has dissolved.
4. If the DNA is <300bp add 250uL of isopropanol
5. transfer into the Zymo column inside a collection tube (small clear guys)
6. spin through, discard waste.
7. Add 200 uL of PE buffer (which is basically 70% ethanol)
8. spin through, discard waste.
9. Add 200 uL of PE buffer
10. spin through, discard waste.
11. spin for 90 seconds, full speed to dry.
12. elute with 8.5 uL of water into a fresh Eppendorf tube

EcoRI/BamHI Digest of PCR Products

For PCR products, you will only digest a portion of your purified PCR product. Note that you must make a minor modification to the procedure if your DNA is shorter than 300bp (you add a little isopropanol to the ADB after melting).

• Set up the following reaction:

 8uL of eluted PCR product
 1uL of NEB Buffer 2
 0.5uL EcoRI
 0.5uL BamHI

• Incubate at 37 degrees on the thermocycler for 1hr
• Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point
• If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column

Run Gel then Zymo Gel Purification

• All spins until the drying step are 15 second full speed spins.

1. cut out bands minimizing extra gel matter.
2. put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
3. heat at 55, shake and/or vortex until the gel has dissolved.
4. If the DNA is <300bp add 250uL of isopropanol
5. transfer into the Zymo column inside a collection tube (small clear guys)
6. spin through, discard waste.
7. Add 200 uL of PE buffer (which is basically 70% ethanol)
8. spin through, discard waste.
9. Add 200 uL of PE buffer
10. spin through, discard waste.
11. spin for 90 seconds, full speed to dry.
12. elute with 8.5 uL of water into a fresh Eppendorf tube