Preparation of Custom Synthesized RNA Transcript Standard
Briefly spin the tube to ensure the contents are on the bottom.  
Resuspend the sample in a volume of TE Buffer resulting in a stock concentration of 0.1 ug/uL.  
To prepare a working solution add 1 uL of the stock solution to 99 uL of water to reach a concentration of 1.0 ng/uL.  
Store resuspended DNA at -20 °C. 
Place frozen competent cells and pre-labeled tubes on ice. 
Pre-warm a hot water bath to 42 °C. 
In a tube on ice add 2 uL (~2 ng) of plasmid working solution to 100 uL of thawed competent cells, flick the tubes to gently mix.  
Incubate the mixture on ice for 30 minutes.  
Heat shock the tubes for 45 seconds in the 42 °C bath. 
Immediately place the tubes on ice for 2 minutes.  
Add 500 uL of SOC or LB to each tube and incubate the tubes at 37°C for 1 hour with shaking (~225 rpm).  
Pre-warm agar plates to 37°C in incubator.  
Take 10 uL, 100 uL, and 200 uL and spread on LB-Amp agar plates.  
Place the plates upside down in 37°C incubator for 12-24 hours.  
Inoculate a 5 uL liquid LB-Amp culture by picking a single well-isolated colony from the LB-Amp plate grown overnight.  
Grow liquid culture at 37°C for ~8 hours with vigorous shaking (~300 rpm).  Remove 850 uL of starter culture and place into a 2 mL freezer vial with 150 uL of sterilized 100% glycerol, mix thoroughly, and store at -80 °C.  
Dilute the starter culture 1/500 to 1/1000 into a larger volume of LB-Amp.  
Grow the culture at 37°C with vigorous shaking (~300 rpm) for 12-16 hours.  
Harvest the culture by centrifugation at 6000 x g for 15 min at 4°C.  
Discard supernatant.  
Recover plasmid DNA using a commercial plasmid mini-prep and the corresponding protocol. 
In a PCR tube combine in the order listed:
12.8 uL Nuclease-free water
2.00 uL NEBuffer 4 
(10X)0.20 uL BSA 
(100X)4.00 uL Plasmid DNA (0.5 ug/uL). 
In a thermocycler incubate the mixture for 1 hour at 37 °C. 
Inactivate the enzyme by incubating at 65 °C for 20 minutes. 
Bring the reaction to 100 uL by adding TE buffer.  
Add 100 uL of Phenol:Chloroform:Isoamyl alcohol (25:24:1).  
Vortex Centrifuge for 5 minutes at 11,000 rpm.  
Transfer the aqueous phase to a new tube.  
Add 0.1 volumes (10 uL) of 3M Sodium Acetate and 0.7 volumes (70 uL) of Isopropanol to the aqueous phase.  
Mix and incubate for 10 minutes at room temperature. 
Spin for 30 minutes at 11,000 rpm at 4 degrees Celsius.  
Discard the supernatant.  
Wash the pellet with 200 uL of ice cold 70% ethanol.  
Centrifuge for 5 minutes.  
Air dry the pellet.  
Resuspend in 5 uL of nuclease-free water.  
Transfer 2 uL of linearized plasmid prep into a new tube.  
Add 2 uL sterile water to the 2uL sample.  
Use 1 uL of the diluted sample to check the concentration of the plasmid prep.  
Run remaining 3 uL of diluted plasmid prep on a 1% agarose gel to check for complete digestion. 
In a tube combine the following at room temperature (Ambion MEGAscript T7 kit): Mix Thoroughly by flicking. 
Incubate mixture at 37 degrees Celsius for 16 hours. 
Add 1 uL Turbo DNase to a concentration.  
Incubate for 15 minutes at 37° Celsius.  
Extract with 1 volume of citrate-saturated (pH 4.7) phenol:chloroform:isoamyl alcohol (25:24:1).  
Vortex for 1 minute. 
Centrifuge for 2 minutes at 12,000 x g. 
Transfer the upper, aqueous phase to a fresh tube.  
Add 1 volume chloroform:isoamyl alcohol (24:1).  
Vortex for 1 minute. 
Centrifuge for 2 minutes at 12,000 x g. 
Transfer the upper aqueous phase to a fresh tube.  
Add 0.1 volumes of 3.0 M sodium acetate and 0.7 volumes of isopropanol.  
Mix and incubate at room temperature for 10 minutes.  
Centrifuge for 30 minutes at 4 °C. 
Carefully discard supernatant.  
Wash pellet with 200 uL of 70% ethanol.  
Dry the pellet under vacuum.  
Resuspend the RNA pellet in 50 uL of water.  
Store at -70° Celsius.  
Quantify RNA using nanodrop.  
Check transcript size using Experion/Bioanalyzer
