Template:SBB-SOEing

Full SOEing Run

Here is the complete SOEing reaction guide from the initial PCR from genomic DNA through Plating.

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.

Protip Make sure you try and get your concentrations and time intervals correct on this step. You may get unwanted activity.

Run Gel then Zymo Gel Purification

Protip For the gel:
1. Use 4ul of the ladder. The ladder run length can be found here. As a good rule of thumb, the first strongly glowing band is 5k, followed by 1.5 and finally 500kb.
2. Run for 5-10 minutes depending on your gel size. Make sure to add loading dye at the proper dilution etc depending on your loading dye. The current loading dye is 6x. That means for 1 ul of the loading dye, you need 5ul of your dna. As an example for class: you want to use 10 ul of your dna and 2 ul of the loading dye (6x)
3. View the gel under the TREE FORT in the corner of the lab.

• 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

Protip Also sufficient is 30 seconds at 14k rpm for the initial spins.

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

Protip Make sure you recalculate the side of the band as it should increase from the first round. A link to the construction files is here.

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

Protip For the gel:
1. Use 4ul of the ladder. The ladder run length can be found here. As a good rule of thumb, the first strongly glowing band is 5k, followed by 1.5 and finally 500kb.
2. Run for 5-10 minutes depending on your gel size. Make sure to add loading dye at the proper dilution etc depending on your loading dye. The current loading dye is 6x. That means for 1 ul of the loading dye, you need 5ul of your dna. As an example for class: you want to use 10 ul of your dna and 2 ul of the loading dye (6x)
3. View the gel under the TREE FORT in the corner of the lab.

• 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

Protip 30 seconds at 14k rpm is also sufficient for the initial spins.

EcoRI/BamHI Digest of PCR Products and Vector

Protip On this step you are digesting both the vector and product. Gel purify both. This step and the follow gel purification steps are for both the product and vector.

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

Protip For the gel:
1. Use 4ul of the ladder. The ladder run length can be found here. As a good rule of thumb, the first strongly glowing band is 5k, followed by 1.5 and finally 500kb.
2. Run for 5-10 minutes depending on your gel size. Make sure to add loading dye at the proper dilution etc depending on your loading dye. The current loading dye is 6x. That means for 1 ul of the loading dye, you need 5ul of your dna. As an example for class: you want to use 10 ul of your dna and 2 ul of the loading dye (6x)
3. View the gel under the TREE FORT in the corner of the lab.

• 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

Protip 30 seconds at 14k rpm is also sufficient for the initial spins.

Ligation of EcoRI/BamHI digests

Protip Make sure you get the timing and concentration as correctly as possible. You don't want unwanted activity.

• 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

Transformation by heat-shock then Plate

Protip On this step, the concentrations are not critical. The purpose of KCM is to change the membrane potential of the cells such that the membrane potential of the cell matches that of DNA and allows for DNA to enter the cell. KCM are just salts of potassium, chlorine, and magnesium.

Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.

  1. Thaw a 200 uL aliquot of cells on ice
  2. Add 50 uL of water to the cells
  3. Add 30 uL of KCM to the cells 
  4. Put your ligation mixture on ice, let cool a minute or two
  5. Add 70 uL of the cell cocktail to the ligation, stir to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
 10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight