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# Goal/Experiment:
The goal of this experiment is to perform 'around-the-horn' or 'divergent' PCR, where primers extend in opposite directions on a plasmid to generate a linear product.

# Around-the-horn PCR and Cloning
*Stephen Floor*

## Abstract
This protocol is designed for 'around-the-horn' or 'divergent' PCR, where primers go around most or all of a plasmid but are pointed away from each other so they generate a linear product. Note that this protocol is written for Q5 polymerase but works fine with other polymerases. To switch polymerases, just change the PCR reaction setup.

**Citation:** Stephen Floor Around-the-horn PCR and cloning. [protocols.io](https://dx.doi.org/10.17504/protocols.io.rf2d3qe) doi:10.17504/protocols.io.rf2d3qe  
**Published:** 03 Jul 2018

## Before start
Program the thermocycler with the PCR program in the main protocol.

## Materials
- **Q5 Hot Start High-Fidelity DNA Polymerase** - 500 units (M0493L) by New England Biolabs
- **Phusion Hot Start Flex DNA Polymerase** - 100 units (M0535S) by New England Biolabs
- **HotStart ReadyMix (KAPA HiFi PCR kit)** (KK2601) by Kapa Biosystems
- **dNTP** (639125) by Takara
- **Forward primer (25 µM)** by Contributed by users
- **Reverse primer (25 µM)** by Contributed by users
- **Template** (5 ng/µl) by Contributed by users

## Protocol

### Design Primers
The basic idea of this protocol is that primers head 'away' from each other on a plasmid backbone, which gives a lot of flexibility in what can be done. Downsides are that it generates a linear product instead of a circular one, so it must be phosphorylated and ligated, and the PCR process takes a while.

Insertion (region in blue are inserted):  
![Insertion Diagram](img_insertion)
 
Deletion (region in red is deleted):  
![Deletion Diagram](img_deletion)

Note that deletions and insertions can be combined. This strategy can be helpful when designing products for Gibson cloning.

### Set up the PCR
**Step 1.**  
Mix the following on ice:

| Reagent                   | Volume for 1 reaction |
|---------------------------|-----------------------|
| Q5 buffer                 | 10 µl                 |
| dNTPs (10 mM)             | 1 µl                  |
| Forward primer (25 µM)    | 1 µl                  |
| Reverse primer (25 µM)    | 1 µl                  |
| Template (5 ng/µl)        | 5 µl                  |
| Q5 polymerase             | 0.5 µl                |
| ddH₂O                     | 31.5 µl               |

### Run the PCR
**Step 2.**  
Run this PCR:

- 95°C for 2 minutes
- 95°C for 15 seconds
- 65°C for 15 seconds
- 72°C for 10 minutes

Repeat the above 30 times

- 72°C for 15 minutes

Note that the extension times at 72°C can be adjusted to the plasmid. In general, allow 1 minute for each kb of plasmid.

### Remove the Template DNA
**Step 3.**  
This strategy will have high background unless you remove the template DNA. DNA from most *E. coli* strains is methylated and can be degraded by the relatively nonspecific restriction enzyme Dpn1.

Add 1 µl Dpn1 to each PCR tube and incubate for 30 minutes to overnight at 37°C.

### Purify the PCR Product
**Step 4.**  
In general, it is recommended to gel purify PCR products from these reactions both to further get rid of template DNA and to avoid cloning any truncated PCR products.

**Step 5.**  
Pour a 1% agarose gel in 0.5X TBE.

- 75 ml 0.5X TBE
- 750 mg agarose

Mix and microwave until boiling and clear - about 90 seconds  
Mix and check that the agarose is dissolved.  
Add 7.5 µl SYBR safe

**Step 6.**  
Casting gel  
Assemble the gel cassette with combs. Use combs that are big enough to accommodate the entire PCR. Typically, these have four or five lanes per gel. Can use two combs per gel.  
Pour hot agarose into cassette and let cool to RT.

**Step 7.**  
Running gel  
Put RT gel into a tank with 0.5X TBE  
Ensure gel is submerged in TBE.

Load ladder into one well. Typically, 10 µl ladder is sufficient, even in large lanes.  
Load samples into remaining lanes.  
Run gel at 120V for 30 minutes

**Step 8.**  
Cut PCR product bands  
Image gel on the blue light imager  
Prepare one 1.5 ml tube for each successful band  
Cut band out with a clean razor blade and transfer to tube.

**Step 9.**  
Gel purify the PCR product according to a mini-spin protocol, eluting in 15 µl. Quantify the product using a nanodrop. Good yields are 50 ng/µl; often yields are 10 ng/µl (which can still work).

### Phosphorylate, Ligate, and Transform
**Step 10.**  
Ligation of unphosphorylated DNA can be accomplished by simultaneous phosphorylation using T4 PNK and ligation with T4 DNA ligase. The reaction setup is simple but ensure you use T4 DNA ligase buffer and **not** T4 PNK buffer, since PNK buffer has no ATP.

**Step 11.**  
**Phosphorylation**  
Mix the following in a tube:  
- 1 µl 10X T4 ligase buffer
- 1 µl PNK
- 50 ng of gel purified PCR product
- Water to 9 µl

Incubate for 30 minutes at 37°C

**Step 12.**  
**Ligation**  
Move to RT, add 1 µl T4 DNA ligase  
Incubate for 1 or more hours at RT (1 hour typically sufficient)

**Step 13.**  
**Transformation**  
Thaw competent cells from -80°C on ice  
Add 4 µl reaction to 33 µl competent cells in a microfuge tube  
Incubate on ice for 25 minutes  
Heat shock at 42°C for 1 minute  
Incubate on ice for 2 minutes  
Add 180 µl LB or SOC media  
Shake at 37°C for 1 hour  
Plate 75 µl on a plate with proper antibiotic

**endofoutput**
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