How do I simulate inverse PCR with a circular plasmid?
This lesson show how to simulate Inverse PCR and recircularization by blunt end ligation.
An equivalent procedure, using overhang-based fusion/recircularization (Gibson or NEBuilder methods) can also be simulated in SnapGene. For more details, see Circularize a Fragment by Gibson Assembly® and Circularize a Fragment by NEBBuilder® HiFi DNA Assembly.
Use Inverse PCR to Amplify/Linearize an Entire plasmid
Choose the Linearization Point
Open the circular plasmid sequence that will be used as template for the PCR.
Switch to "Sequence view".
- Click within the sequence to set the point for linearization.
- Note the coordinate of the nucleotide to the right of the insertion point (in this example position 470). This nucleotide will be the first nucleotide of the linearized plasmid.
- Note the coordinate of the insertion point (in this example position 469). The nucleotide to the left of the insertion point will be the last nucleotide of the linearized plasmid.
- Click Edit → Select Range.
In the "Range Selection" dialog, enter the coordinate of the first nucleotide in the leftmost field.
Enter the coordinate of the first nucleotide in the left field.
Enter the coordinate of the insertion point (last nucleotide) in the right field
Enter the Confirm the predicted fragment size (in this example 2686 bp) matches the size of the complete circular plasmid.
Click Select to select the entire sequence, starting at the desired linearization point.
Skip to the section "Open the PCR Dialog".
Option 2: Use Inverse PCR to Amplify/Linearize a Portion of a Plasmid
Select the Region to Remove
In Map view, click on a feature to select it for removal, or in Sequence view, select a specific sequence region for removal.
To invert the selected region, click Edit → Invert Selection.
The selected region will be amplified by Inverse PCR.
Open the PCR Dialog
To open the PCR dialog, click Actions → PCR....
Choose the Primers
To automatically design primers to amplify the select region, set the desired Tm and click Choose Primers.
Create the PCR Product
In the PCR dialog:
- Revise and add appropriate primer names.
- Optional: Edit the primer 5'–ends if you want to add short sequences to the PCR product ends.
- Review the phosphorylation state. Add a phosphate group to at least one primer by clicking one of the 5' Phosphorylated check boxes. If checked these indicate you plan to either a) use phosphorylated primers for the PCR or b) phosphorylate the amplified PCR product with DNA kinase.
- Set the Polymerase: to Creates Blunt Ends if you plan to amplify with a polymerase that will generate ends suitable for blunt end ligation.
- Name the PCR product.
Click PCR to create the "Inverse PCR" product.
View the Fragment
The linearized fragment will be created as a new unsaved file.
Click Save to save the new PCR product sequence to an appropriate location on your computer.
Circularize the Fragment
To circularize the fragment, click Actions → Circularize....
Name of the circularized vector and then click Circularize.
View the Circularized Product
The circular product will be created as a new unsaved file.
Click Save to save the new sequence to an appropriate location on your computer.
