Design and preparation of DNA IVT template¶
We currently have two harbors (pHAs) for IVT: one contains human beta globin 5’ and 3’ UTRs (pKG01864) and the other contains the 5’ and 3’ UTRs from the Pfizer-BioNTech COVID-19 vaccine (pKG01834).
Important
As of 2023.06.04: use pKG01864. The Pfizer-BioNTech template (pKG01834) contains two point mutations in the 3’ UTR, presumably from using 293T genomic DNA for cloning. We’ve witnessed poor expression in primary cells (both MEFs and human adult fibroblasts) relative to the beta-globin UTRs. If anyone would like to fix these and characterize (undergrad project?), that could prove useful!
Creating a new IVT template plasmid¶
As a pHA, pKG01864 is compatible with existing pPV2s for BsaI golden gate cloning.
If a desired CDS is not available, you can either 1) make a pPV2 or 2) clone directly into pKG01864 via Gibson. If proceeding with option 2, the plasmid backbone can be PCR amplified with the following primers:
VECTOR-FWD: 5’-ctcgagctcaagcttcgaattcac-3’ VECTOR-REV: 5’-gaccggtagcgtgcttt-3’
Generating IVT linear DNA template from an IVT template plasmid¶
Our IVT platform relies on PCR amplification using a universal primer pair and a plasmid harboring the CDS of interest as template to generate the IVT template. The forward and reverse primers bind to the 5’ and 3’ beta globin UTR, respectively. The forward and reverse primers also encode the T7 polymerase promoter and the polyA tail, respectively.
Note
Alternatively, one could encode these elements on the plasmid and generate IVT template via restriction digestion. Although obviating the need for PCR and using an expensive oligo, the downside of this method is variable polyA tail length due to truncation of long repeat A sequences in E. coli. There is a report of reducing these recombination events, if someone is feeling ambitious and would like to build/test this :)
If a PCR-generated linear template already exists, skip to step 4. Else, perform a small-scale PCR on a new IVT template to verify desired amplification and product specificity:
Component |
Amount |
|---|---|
5x Q5 buffer |
5 |
dNTP mix |
0.4 µL |
CleanCap AG F-primer (10 µM) |
0.2 µL |
R-primer (10 µM) |
0.2 µL |
Plasmid Template DNA (1 ng/µL) |
1.0 µL |
Q5 polymerase |
0.1 µL |
Water |
to 20 µL |
Important
Make sure you’re using the correct primer pairs for a given plasmid IVT template backbone!
Perform PCR using 57 C annealing temperature, adjust extension time based on length of linear template.
Cleanup the PCR product and run an aliquot on a gel to validate the desired product size was amplified.
Note
This template can now serve as a “master” PCR template for future PCR amplifications. This is advantageous for tricky PCR amplicons and essential for those that contain internal primer binding sites (such as pKG02007, which harbors Cas9), as the annealing temperature can be increased due to the lengthened primer/template homology.
Perform the PCR outlined in step 1, substituting the Plasmid Template DNA with the PCR-generated linear template DNA. Scale-up accordingly to generate the amount of linear IVT template needed for your IVT reaction. Store unused linear PCR template in the IVT box and use as needed.