TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is from:<br /><br />Satinsky, Brandon M., et al. "<a href="http://www.sciencedirect.com/science/article/pii/B9780124078635000125" target="_blank">Use of internal standards for quantitative metatranscriptome and metagenome analysis.</a>" Methods in enzymology 531 (2012): 237-250.<br /><br />Please see the <a href="http://www.sciencedirect.com/science/article/pii/B9780124078635000125" target="_blank">full chapter</a> for additional details.

[GUIDELINES]
<strong>Required materials<br /><br /></strong>• Equipment: 4 degrees Celsius microcentrifuge, 10-, 20-, 200-, and 1000-uL pipettes, water bath, 37 degrees Celsius shaking incubator, thermocycler, gel electrophoresis equipment and reagents, microfluidic electrophoresis instrument or fluorometry-based instrument for measuring nucleic acid concentration. <br /><br />• Media: LB agar, LB agarþampicillin (100 ug/mL final concentration), LB medium, LB mediumþampicillin (100 ug/mL final concentration), SOC medium (2% tryptone, 0.5% yeast extract, 10 mM sodium chloride, 10 mM magnesium sulfate, 20 mM glucose).<br /><br />• Bacterial cell line: One Shot® Top10 Chemically Competent Escherichia coli (Life Technologies, Grand Island, NY).<br /><br />• TemplateDNA: Custom synthesizedDNAtemplate (T7 RNA polymerase promoter, internal standard sequence, unique restriction site) inserted into a plasmid.<br /><br />• Restriction digest and end repair: Restriction enzyme matching unique restriction site and corresponding buffers, mung bean nuclease for end repair on digests that do not produce blunt ends. <br /><br />• Commercially available kits: Ambion MEGAscript® T7 Kit (Life Technologies), Quant-iT™ RiboGreen® RNA Assay Kit (Life Technologies), miniPrep plasmid extraction kit.<br /><br />• Other reagents: phenol:chloroform:isoamyl alcohol (24:24:1, pH 7), citrate-saturated phenol:chloroform:isoamyl alcohol (24:24:1, pH 4.7), sterile 2-propanol, ice cold 70% ethanol, nuclease-free 3M sodium acetate, nuclease-free TE buffer, sterilized 100% glycerol; 1% agarose gel, nuclease-free water, RNaseZap® (Life Technologies).<br /><br />• Disposables: nuclease-free 10-, 20-, 200-, and 1000-uL filter tips, nuclease-free PCR tubes, nuclease-free microcentrifuge tubes, gloves.<br /><br /><strong><br />Plasmid amplification and stock preparation</strong><br /><br /><strong>Resuspension of plasmid DNA</strong><br />If beginning with lyophilized plasmid DNA, spin briefly to ensure the contents are at the bottom of the tube. Resuspend the plasmid DNA in a volume of TE buffer to produce a stock concentration of 0.1 ug/uL. To prepare a working solution, add 1 uL of the stock solution to 99 uL of nuclease-free water to produce a final concentration of 1 ng/uL. The resuspended plasmid DNA can be stored at -20 degrees Celsius.<br /><br /><strong>Chemical transformation of plasmid into Top10 E. coli cells</strong><br />Prior to beginning the transformation, ensure that all required media are prepared and sterilized. Place frozen competent cells and a prelabeled tube on ice. Prewarm a hot water bath to 42 degrees Celsius. To a tube on ice, add 2 uLof (~2 ng) the plasmid working solution to 100 uL of thawed competent cells and flick the tube gently to mix. Incubate the mixture for 30 min on ice, then heat shock in the 42 degrees Celsius hot water bath for 45 s. Immediately place the tube on ice for 2 min and then add 500 uL of SOC or LB liquid medium to the tube and incubate at 37 degrees Celsius for 1 h with shaking (~225 rpm). During this time prewarm LB-Amp agar plates in a 37 degrees Celsius incubator. From the tube, pipet and spread 10, 100, and 200 uL on three separate LB-Amp agar plates. Place the plates upside down in a 37 degrees Celsius incubator for 12â24 h. Following incubation, inoculate a single, well-isolated colony from one of the plates and place into 5-mL LB-Amp media. Grow the liquid culture at 37 degrees Celsius for ~8h with vigorous shaking (~300 rpm). Remove 850 uL of the starter culture and place into a 2-mL freezer vial with 150 uL of sterilized 100% glycerol, mix thoroughly, and store at -80 degrees Celsius. To work from the frozen stocks, place a loopful of stock into 10 mL of LB-Amp liquid medium and grow at 37 degrees Celsius with vigorous shaking (~300 rpm) for 12â16 h. Harvest cells by centrifugation at 6000 x g for 15 min at 4 degrees Celsius. Discard the supernatant and recover the plasmid DNA using a commercially available plasmid mini-prep kit.<br /><br /><strong>Plasmid linearization and in vitro transcription</strong><br /><strong><br />Linearization of plasmid template </strong><br />Digest 2 ug of plasmid with restriction enzyme targeting the site at the end of the template sequence according to the restriction enzyme protocol. Sticky ends created by nonblunt-end cutting enzymes should be removed using mung bean nuclease. After digestion and end repair, bring the reaction to 100 uL by adding TE buffer, add 100 uL of phenol:chloroform:isoamyl alcohol (25:24:1, pH 7), and mix by vortexing. Spin the mixture for 5 min at 12,000 x g. Transfer the aqueous phase to a new tube and add 0.1 volumes (10 uL) of 3M sodium acetate and 0.7 volumes (70 uL) of isopropanol to the tube.Mix thoroughly and incubate for 10 min at room temperature, and centrifuge for 30 min at 12,000 x g at 4 degrees Celsius. Discard supernatant and wash pellet with 200 uL of ice cold 70% ethanol. Centrifuge for 5 min and discard the supernatant, being careful not to disturb the pellet. Air-dry the pellet to remove residual ethanol before resuspending the pellet in 5 uL of nuclease-free water. Transfer 2 uL of linearized plasmid into a newtube and add 2 uL of nuclease-free water. Use 1 uL of the diluted sample to check the concentration and analyze the remaining 3 uL on a 1% agarose gel to check for complete digestion and the presence of a single-sized product. Retain the 3 uL of undiluted DNA template for subsequent steps.<br /><br /><strong>Synthesis and purification of mRNA internal standard</strong><br />Synthesis of the internal standards from a template containing a T7 promoter is completed through the use of an in vitro transcription reaction using the Ambion MEGAscriptÂ® High Yield T7 Kit. In a 0.2-mL tube at room temperature, combine 2 uL of ATP solution, 2 uL of CTP solution, 2 uLof GTP solution, 2 uL of UTP solution, 2 uL of 10x reaction buffer, 1 ug of linearized template DNA (up to 8 ul), and 2 uL of enzyme mix, and bring the total reaction volume to 20 uL with nuclease-free water. Mix thoroughly by flicking and incubate themixture at 37 degrees Celsius in a thermocycler with a heated lid for 16 h. Degrade the plasmid DNA by adding 1 uL of Turbo DNAse to the reaction tube and incubating for 15 min at 37 degrees Celsius. Add 20 uL of citrate-saturated (pH 4.7) phenol:chloroform:isoamyl alcohol (25:24:1) to the tube. Vortex the mixture for 1 min and centrifuge for 2 min at 12,000 x g to separate the phases. Transfer the upper aqueous phase to a fresh tube and add 1 volume of chloroform:isoamyl alcohol (24:1). Vortex the mixture for 1 min and centrifuge for 2 min at 12,000 x g. Transfer the upper aqueous phase to a fresh tube and add 0.1 volumes of 3M sodium acetate and 0.7 volumes of isopropanol. Mix by vortexing and incubate for 10 min at roomtemperature and then centrifuge for 30 min at 4 degrees Celsius. Carefully discard the supernatant and wash the pelletwith 200 uL of ice cold 70% ethanol. Centrifuge for 5 min and carefully remove the supernatant without disturbing the pellet. Air-dry the pellet until no residual ethanol remains and resuspend the dried pellet in 50 uL of nuclease-free water. Quantify the RNA fluorometrically using a QuantiTâ¢ RiboGreenÂ® RNA Assay Kit and check the transcript size using a microfluidic electrophoresis instrument (e.g., Experion Automated Electrophoresis System, Agilent 2100 Bioanalyzer, or Agilent 2200 TapeStation). Store the mRNA internal standard stock at -80 degrees Celsius.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.