Preparation of Encoding Probes SOP005.v1.5 (PCR, In-vitro Transcription, Reverse Transcription and USER ENZYME Digest)

In a 1.7 mL Eppendorf tube, mix the following:

• 40µL ;

•  2µL ;

•  2µL ; 

• 1µL ; 

•  355µL ; 

•  400µL .

Aliquot 25µL into 32 PCR tubes.

Run the following protocol on a qPCR machine:

1. 98°C for 0h 0m 30s;

2. 98°C for 0h 0m 10s;

3. *63°C for 0h 0m 10s;

4. 72°Cfor 0h 0m 15s;

5. Measure the fluorescence of each sample. 

*Adjust temp for the specific primer pair.

Repeat cycle steps 2 through 5 from step 3 until the rate at which the sample amplification decreases and starts to reach a plateau.

Remove samples during the 72°C.  Since the oligo pool consists of a complex number of sequences, samples removed outside of the elongation step will likely lead to hybridization with complement mismatches. The simplest method would be to run 10-14 rounds of PCR amplification and allow the instrument to complete the final round. If it is uncertain how many rounds you need to run for a particular oligo pool, you can set the instrument for an estimated number of rounds and then stop the round at any 72°C elongation step.

Column purify to remove enzyme, nucleotides, and primers.

In a 15 mL Falcon tube, mix the following:

800µL generated in Step 5 ;

4mL .

Run this mixture across a 100-µg capacity column using either a vacuum manifold.

Wash the column twice with 300µL , spinning the column in a table top centrifuge at maximum speed for 0h 0m 30s each time.

Elute the template by adding 170µL to the column, transferring the column to a fresh 1.7 mL Eppendorf tube, and spinning at maximum speed for 0h 0m 30s.

Set aside 10µL for quality control.

Two quality control steps can be performed to verify the quality of your PCR product.

1. Using a spectrophotometer, such as the Nanodrop or similar, measure the concentration of dsDNA in your product. The concentration should be between 0.01µg/µL to 0.05µg/µL.

2. The second quality control step is gel electrophoresis and will be described in part 4, step 5 (>>step 36 below).

The second step of this protocol is a high yield in vitro transcription reaction that further amplifies the template molecules created in Part 1 and converts them into RNA.

In a fresh 1.7 mL Eppendorf tube, mix the following:

160µL created in Part 1 .

176µL

250µL provided with the Quick HiScribe T7 polymerase kit

25µL

25µL (from the same HiScribe kit)

Incubate the reaction in a 37°C incubator or dry bath for 12h 0m 0s–16h 0m 0s hours. While the reaction is complete after 6-8 hours, it is convenient to leave this reaction overnight. Remove 20µL for quality control.

To confirm that the in vitro transcription was successful, column purify the reaction then measure its concentration with a spectrophotometer.  

To purify, mix the following:

• 20µL

• 30µL

• 100µL

• 150µL . 

Pass across a 100-ug-capacity spin column in a table-top centrifuge.

Wash this column once with 400µL , centrifuge 0h 0m 30s top speed.

Wash twice with 200µL , centrifuge 0h 0m 30s top speed.

Elute the RNA with 100µL .

If successful, the concentration of the in vitro transcription should be between 0.5µg/µL to 2µg/µL.

Purified RNA can also be run on a gel as described in Part 4 , Step 5 (>>step 36 below).

In this step of the protocol, the large quantities of RNA produced by the high yield in vitro transcription are converted to single-stranded DNA using a reverse transcription reaction. To cleave off the 5' priming region and shorten the final probe, we use a uracil-modified reverse transcription primer for the reverse transcription reaction followed by use of a Uracil-Specfic Excision Reagent (USER Enzyme) to cleave the uracil nucleotide and remove the primer. This RNA templates are then removed via alkaline hydrolysis, and the final encoding probes are purified and concentrated.

To the unpurified in vitro transcription created in Part 2 , add the following and mix well:

• 200µL

• 120µL with uracil modification.

• 240µL

• 24µL

• 24µL .

Incubate in a 50°C for 1h 0m 0s hour. It is important to use a water bath, not an air incubator, to ensure that the temperature of the sample rises to 50°C quickly.

To digest, use a 1:20 (vol/vol) and incubate at 37°C for 24h 0m 0s to cleave off the priming region at the site of the uracil.

Split the above reaction into two 1.7-mL Eppendorf tubes and add the following to each:

•  300µL

• 300µL

Incubate in a 95°C for 0h 15m 0s.

Combine the two aliquots above into a single 50 mL Falcon tube and add the following:

• 4.8mL

• 19.2mL .

Mix well and split equally between eight 100-µg capacity spin columns.

Pull the sample across the columns with a vacuum manifold or via centrifugation.

Wash the columns once with750µL.  Centrifuge to remove all of the wash buffer. You may need to centrifuge this twice to fully remove all of the wash buffer.

Elute the columns using 100µL .

Combine eluates and set aside 10 µL for quality control.

Use a vacuum concentrator to dry the samples.  This process could take several hours.  Re-suspend the dried pellet in 24µL or if desired, hybridization buffer.  Store probe at -20°C and avoid unnecessary Freeze-thaw cycles.

If vacuum concentrator isn’t available, concentrate probe using ethanol precipitation (refer to SOP009).

Quality Control. Analyze the concentration and the purity of your finalized probe sample on a Nanodrop spectophotometer or similar. Your ssDNA concentration should be around 34µg/µL.

We suggest running your PCR product, IVT product and your final probe product on a 15% TBE-urea polyacrylamide gel to review the size and quality of your products. Smearing can indicate degradation of your product, particularly for RNA molecules. You should also note the size of the bands your are expecting to see and compare each band to one another. IVT will shorten your product by 20nt removing the 3' primer as the reaction only occurs after the T7 promoter region at the 3' end of the probe. Additionally, the USER enzyme digested product should be approximately 20nt shorter as this removes the 5' priming region. These changes in the product lengths should be visible when running your gel.