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From 2011.igem.org

Contents 1 Protocol 1.1 Cloning 1.2 Binding assays 1.2.1 Ligation 1.2.2 Restrictive Digestion 1.3 Medium 1.3.1 Medium for drosophila 1.3.2 M9 medium 1.3.3 LB medium 1.3.4 SOB medium 1.3.5 SOC medium 1.3.6 Buffer TB 1.3.7 DNS reaegnt 1.4 Measurement 1.4.1 Solubilization of Antibiotics 1.4.2 Transformation 1.4.2.1 Sequence 1.4.2.2 Measurement of RPU

Protocol

Cloning

Binding assays

Ligation

1. Make 2µL of Mixture (the vector and the insert at 1 : 5-10) and Control (only the vector).
2. Add 5µL Ligation High, 1µL T4 Kinase, and 7µL MilliQ to create a solution.
3. Incubate at 16℃ for 30 min. If the colonies of E.coli transformed with the Control

Restrictive Digestion

1. Use EcoRI, XbaI, SpeI, PstI, (NEB)
2. Mix the following.

   Sample	5µL
   10xBuffer	1µL
   Restriction Enzyme	0.1µL
   MilliQ	-3.9µL

3. Let stand for 2h at 37℃

Medium

Medium for drosophila

1. Stir dry yeast and agarose with about two-thirds of water. Then, autoclave it.
2. Stir corn flour and glucose with the remaining water.
3. Stir 1 and 2, then autoclave it again.
4. after autoclave, add propionic acid and 10 % p-hydroxybenzoate in 70 % Eternol into it.

[http://www.biol.se.tmu.ac.jp/fly/www/standard-medium.html Medium for drosophila]

M9 medium

1. Stir Na2HPO4 6 g, KH2PO4 3 g, NaCl 0.5 g and NH4Cl 1 g with water 1 l.
2. After autoclave, add 10 ml filter sterilized 100 mM MgSO4, 20 % glucose, 10 mM CaCl2, 100 mM thiamine-HCl.
3. If you need, add 10 ml filter sterilized 20 % casamino acid.

LB medium

1. Stir Tryptone 20 g, Yeast extract 10 g and NaCl 10 g with water 200ml.
2. If you make LB plates, add agar 10 g.
3. Autoclave.

SOB medium

1. Stir Tryptone 20g and Yeast extract 5g with water.
2. Add 2 ml 5 M NaCl and 840 ul 3 M KCl and add water up to 1 l.
3. After autoclave, add 10 ml filter sterilized 1 M MgSO4 and 1 M MgCl2.

SOC medium

1. Add 2 M glucose 1 ml to 100 ml SOB.

Buffer TB

1. Stir PIPES and CaCl2・2H2O with 100 ml water.
2. Add 8.315 ml 3 M KCl.
3. Add KOH and adjust pH 6.8.
4. Add MnCl2・4H2O.
5. Add water up to 200 ml.
6. Filter sterilize

DNS reaegnt

1. Add 1% DNS 88ml and Rochelle salt 25.5 g to 4.5% NaOH 30 ml (A solution)
2. Add Phenol 1 g and water 7.8 ml to 10% NaOH 2.2 ml (B solution)
3. Add NaHCO3 6.9g to B solution 6.9 ml
4. Add A solution 118ml to B solution 6.9 ml
5. Leave 2 days
6. Store in brown bottle

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.