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===== Order template =====

===== in vitro transcription =====
==== 1. Annealing Reaction ====
  * Dilution: dilute each DNA strand to a final concentration of 100 µM with water. (# nmol X 10 = µL of water to add)
  * Make the following cocktails in an Eppendorf tube (scale it up according to your final reaction volume)

^ Volume (30 uL in total) ^ Reagent ^
|7.5 uL|DNA strand 1|
|7.5 uL|DNA strand 2|
|9 uL|10 mM MgCl2|
|6 uL|H2O|

  * Incubate the annealing reactions at 95.0 ºC for 5 min, and then immediately put it on ice.

==== 2. in vitro Transcription ====
  * Mix the following reagents in a 50 mL falcon tube
  * Note
    * SP: Spermidine; PEG: Polyethylene glycol
    * RNAP: T7 RNA polymerase (5000 unit)

^ TOTAL ^ H2O^0.5M Tris ^ 0.25M MgCl2 ^ 0.25M DTT ^ 0.25M SP ^ PEG ^ ATP ^ GTP ^ CTP ^ UTP ^ DNA ^ RNAP ^
| 50 uL w/ PEG | 13.8 uL | 10 uL | 6 uL | 6 uL | 0.4 uL | 4 uL | 1.5 uL | 1.5 uL | 1.5 uL | 1.5 uL | 0.8 uL | 3 uL |
| 50 uL w/o PEG | 17.8 uL | 10 uL | 6 uL | 6 uL | 0.4 uL | 0 uL | 1.5 uL | 1.5 uL | 1.5 uL | 1.5 uL | 0.8 uL | 3 uL |
| 9 mL | 3204 uL | 1800 uL | 1080 uL | 1080 uL | 72 uL | 0 uL | 270 uL | 270 uL | 270 uL | 270 uL | 144 uL | 540 uL |
| 12 mL | 4272 uL | 2400 uL | 1440 uL | 1440 uL | 96 uL | 0 uL | 360 uL | 360 uL | 360 uL | 360 uL | 192 uL | 720 uL |
| 15 mL | 5340 uL | 3000 uL | 1800 uL | 1800 uL | 120 uL | 0 uL | 450 uL | 450 uL | 450 uL | 450 uL | 240 uL | 900 uL |

Another：
^ TOTAL ^ 5XTRB ^ T7 RNAP ^ 50μM DNA ^ rNTPs ^ DEPC water ^
| 50μL  | 10μL  |  1.5μL  |  1.5μL   | 1.5μL |   35.5μL   |
5XTRB(Transcription Buffer)：
^        5x 浓度          ^   母液体积  ^  母液浓度  ^  DEPC water  ^
|  200mMTris-HCl pH8.0   |    8 mL    |   1 M     |     ---      |
|  10mM SP               |   1.6 mL   |  0.25 M   |     ---      |
|  5mM DTT               |   0.2 mL   |   1 M     |     ---      |
^  MgCl2梯度:             |    ---     |   ---     |     ---      |
|  50mM                  |    2 mL    |   1 M     |   28.2 mL    |
|  75 mM                 |    3 mL    |   1 M     |   27.2 mL    |
|  100 mM                |    4 mL    |   1 M     |   26.2 mL    |
|  150 mM                |    6 mL    |   1 M     |   24.2 mL    |
|  200 mM                |    8 mL    |   1 M     |   22.2 mL    |
===== Purify RNA =====
==== Option 1: PAGE ====
== Choose proper concentration of PAGE ==
  * 8% Acrylamide: 60~400bp
  * 12% Acrylamide: 40~200bp
  * 15% Acrylamide: 25~150bp
  * 20% Acrylamide: 6~100bp

== Denatured PAGE Gel ==
  * 500V,130mA at 4C for 6~7 hours

== Crush & Soak ==
  * Cut RNA band from the gel under 260nm wavelength fluorescence light
  * Crush the gel band into fine grains by using 50mL Falcon tube and 50mL syringe
  * Soak in 2 volumes of CSB (Crush&Soak buffer) for 6 hours at room temperature, and collect solution by filtering it
  * Repeat the step above
  * Ethanol Precipitate the RNA from the collected supernatant (or run the supernatant through desalting column)
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