How can I insert a PCR product into a Linearized vector?
Some plasmids, for example pminiT 2.0, are sold as linearized fragments ready for ligation to an insert.
This lesson shows how to simulate cloning of a blunt end PCR product into a linearized pminiT 2.0 vector.
Create the PCR Product
Open the target sequence.
In Map view, select the feature to be amplified, or switch to Sequence view and select a specific sequence range.
Click menu Actions → PCR....
Set the desired target Tm for the PCR primers.
SnapGene will design new primers.
- Add appropriate names for the primers.
- Set an appropriate name for the PCR product.
- Set the polymerase to "Create Blunt Ends"†.
- Click PCR to create a new PCR product file.
† The pminiT 2.0 vector is blunted ended so PCR should be be with a polymerase the generates blunt ends.
Click menu File → Save and save the new PCR product file to an appropriate location on your computer.
Open the Vector Sequence
SnapGene provides an online database of commonly used annotated vectors that you can access for your cloning simulations. In this example we obtain the linearized pminiT 2.0 vector sequence from the SnapGene vector database.
Click File → Import → SnapGene Online Sequences... .
Type a search phrase to find the vector.
Select the vector in the list and click Import to download the sequence.
The linearized vector will open in SnapGene.
Click menu File → Save and save the vector file to an appropriate location on your computer.
Ligate the Vector and Insert
Ensure the vector file is in focus, then click Actions → Restriction and Insertion Cloning → Insert Fragment....
The vector will be added to the Vector tab.
If required, click on the vector fragment in the list to define the full length vector fragment as the ligation input.
The vector is linearized so no restriction enzyme should be set.
Switch to the Fragment tab.
Set the "Source of Fragment" as the newly generated PCR product.
If required, click on the insert fragment in the list to define the full length PCR product as the ligation input.
Switch to the Product tab.
A warning will be displayed, this is expected as a blunt end ligation will produce two ligation products.
If you wish to simulate product of both ligation products check the "Alternative product with flipped fragment" option.
Add names for the new product file/s.
Click Clone to create the new files.
View each product file and click menu File → Save and save to an appropriate location on your computer.
