From 2011.igem.org

Protocol

Cloning

Binding assays

Medium

Measurement

Solubilization of Antibiotics

1. Mix the following (Final concentration is 50mg/mL).
   • Ampicillin:

     Ampicillin

     1.0g

     MilliQ

     20mL

   • Kanamycin:

     Kanamycin

     0.5g

     MilliQ

     10mL

2. Dispense 1.1mL of the solution into 1.5mL tubes.
3. Store in the -20℃ freezer.

Transformation

1. Unfreeze conpitent cells on ice.
2. Dry a plate by letting the plate upside down and partly open in incubator.
3. Add 1µL DNA solution and 20µL compitent cells to 1.5mL tube, let stand for 30min on ice. If few colony is observed, increase the amount of the compitent cells or DNA, but make the amount of DNA not to get over that of the compitent cells.
4. Heatshock for 60s at 42℃.
5. Let stand for 2min on ice.
6. Culture for 1h in preculture medium (LB or SOC medium), and plate by using spreader. Do not heat spreader too much because e.coli will dead for heat.

Sequence

1. Use Big Dye Terminator 3.1（ABI）
2. Mix the following

   5xBuffer

   2µL

   Primer (3.2µM)

   1µL

   Template Plasmid 

   200ng

   Big Dye Terminator 3.1

   0.5µL

   MilliQ

   up to 10µL

3. Let stand for 1min at 96℃.
4. 35 cycles for 5s at 98℃, for 5s 50℃, and for 2.5min at 68℃.
5. Add 25µL 100% ethanol and 1µL NAOAC

Measurement of RPU

1. Cultivate E.coli in M9 media(+ casamino acids) for about 15 hours.
2. Dispence 2.4ml to each tube.
3. Centrifuge these tubes (13,000 rpm , 4℃, 1min) and discarded the supernatant.
4. Add 1.2ml media(- casamino acids) and centrifuged at 4℃ twice.
5. Centrifuge these tubes again and discarde the supernatant and added 1.2ml media(-casamino acids) at 37 ℃.
6. Bring up E.coli at 0,5,10,15,20,25,30,60min and extracted RNA and synthesized cDNA.