(Last Updated: March 2020)

The DRSC builds templates the first time using a gene-specific primer with T7 sequence ends. Primers that recognize the T7 sequence can be used for any subsequent re-amplification of the template PCR product. This makes it possible for us to use one primer and set of protocols for templates, regardless of the targeted gene. 

This PCR protocol was adapted from the DRSC/TRiP-FGR site (https://fgr.hms.harvard.edu/dsrna-synthesis). The Phusion® Hot Start High-Fidelity DNA Polymerase (NEB M0535) kit contains the Phusion DNA polymerase and the HF buffer. 

General Notes 

• T7 primers should be diluted to 10 uM. More can be ordered at IDT Primers through the Biopolymers core downstairs. The stock should be diluted to 100 uM, then aliquots of 10 uM can be made. 
• dNTPs should be diluted down to 2.5 mM. The concentration of the stock is 25 uM. 

PCR on Cherry Pick Samples

1)  Remove your cherry pick plates from the -20 freezer. Once thawed, vortex for 30 seconds, then centrifuge for a minute. 

2)  Produce the following master mix for each 96 well plate you wish to amplify. The DRSC uses Phusion® Hot Start High-Fidelity DNA Polymerase; NEB M0535. All the reagents for PCR are located in a cryo box labeled “Kasia PCR” in Freezer #2. 

• Always make several reactions’ worth more of master mix to ensure there is enough. 

3)  Dispense 24 µL of master mix into each well of a 96-well PCR plate. 

4)  Transfer 1 µL of dilute PCR template to its corresponding well. 

5)  Seal the plate using aluminum foil (make sure to use the roller to seal it tight). Mix by inverting. Spin down for 1 minute at 2,000 rpm. 

6)  Run the following PCR program (this protocol is called "PCR-57" on the thermocyclers in Bay 2): 

98°C for 30 seconds 
98°C for 10 seconds 
57°C for 20 seconds 
72°C for 20 seconds 
Repeat steps 2-4 - 35 times 
72°C for 5 minutes 
4°C – for ever 

7)  Analyze 5 µL of the PCR product on a 0.7% agarose gel with a 100bp ladder. Add 1 µL of loading buffer to 5 µL of sample, then load 5 µL of that onto the gel. Check for any blank, smeared, or double bands and note those down. A gradient PCR can be run to attempt to recover those failed reagents. 

8)  Seal the plate with the PlateLoc and store at -20. Make sure all plates are labeled and dated. 

9)  Move onto the IVT protocol. PCR products do not need to be cleaned up for IVT.  

Gradient PCR on Failed Samples (16 samples at a time) 

1)  Produce the above master mix for every 16 amplicons you wish to optimize using different annealing temperatures. 

2)  Dispense 144µL of master mix to wells A1-H2. 

3)  Add 6µL dilute PCR template to its corresponding well. Mix thoroughly by pipetting. 

4)  Using a multichannel pipette, transfer 25µL from column 1 [A1-H1] to column 3 [A3-H3]. Repeat transfer from column 1 to columns 5, 7, 9, 11. 

5)  Using a multichannel pipette transfer 25µL from column 2 [A2-H2] to column 4 [A4-H4]. Repeat transfer from column 2 to columns 6, 8, 10, 12. 

The plate eventually looks like this: 

6)  Seal the plate using aluminum foil. Mix by inverting. Spin down for 1 minute at 2,000rpm. 

7)  Run the following PCR program (this protocol is called "PCR50-65" on the thermocyclers in Bay 2): 

98°C for 30 seconds 
98°C for 10 seconds 
50—65°C for 20 seconds 
72°C for 20 seconds 
Repeat steps 2-4 35 times 
72°C for 5 minutes 
4°C – for ever 

8)  Analyze 5µL of PCR reaction on a 0.7% Agarose Gel with a 100bp ladder. 

9)  Consolidate the best amplicons onto the original “PCR57” plate by replacing the bad amplicons and adding in the successful ones from the gradient plate. Take care to select only the best amplicon on the gradient plate as a replacement. 

• If there are still amplicons that didn’t amplify well at this stage, assuming that they’re only a small fraction of the overall order, it is okay to proceed as if those amplicons are good. Just make sure to write everything down so there are clear records of which reagents were successfully amplified and which ones were not. 

10)  Seal the plate with the PlateLoc and store at -20. Make sure all plates are labeled and dated. 

11)  Move onto the IVT protocol. PCR products do not need to be cleaned up for IVT.