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
