"

From 2011.igem.org

Revision as of 12:46, 19 September 2011 (view source) Sabineoesterle (Talk | contribs) (→Transformation) ← Older edit		Revision as of 12:46, 19 September 2011 (view source) Sabineoesterle (Talk | contribs) (→Ligation) Newer edit →
Line 67:		Line 67:
	# Ligation mix		# Ligation mix
	:* 1:5 ratio of plasmid backbone : Insert		:* 1:5 ratio of plasmid backbone : Insert
-	:* 1 µ 10 x T4 Ligase buffer (vortex)	+	:* 1 µl 10 x T4 Ligase buffer (vortex)
-	:* 0.5 µ T4 Ligase	+	:* 0.5 µl T4 Ligase
	:: adjust to 10 &micro;l with ddH<sub>2</sub>O		:: adjust to 10 &micro;l with ddH<sub>2</sub>O

---

Revision as of 12:46, 19 September 2011

Material and methods	Protocols Plasmid list Primer list
All protocols, plasmids and primers can be found here.

Protocols

Double digest for cloning

	Variant 1	Variant 2
Plasmid	x µl Plasmid (~2 µg if possible) 2 µl EcoRI 2 µl XbaI 5 µl buffer 4 0.5 µl BSA adjust to 10 µl ddH2O	x µl Plasmid (~2 µg if possible) 3 µl PstI 2 µl Spe I 5 µl buffer 2 0.5 µl BSA adjust to 10 µl ddH2O
Insert	x µl Insert (~2 µg if possible) 2 µl EcoRI 3 µl Spe I 5 µl buffer 1 0.5 µl BSA adjust to 10 µl ddH2O	x µl Insert (~2 µg if possible) 3 µl XbaI 2 µl Pst 1 5 µl buffer 3 0.5 µl BSA adjust to 10 µl ddH2O

⇒ incubate for 2 h at 37°C

Ligation

1. Ligation mix

• 1:5 ratio of plasmid backbone : Insert
• 1 µl 10 x T4 Ligase buffer (vortex)
• 0.5 µl T4 Ligase

adjust to 10 µl with ddH₂O

1. Let the mixture stay for 1h at room temperature
2. Denature the ligase at 65 °C for 5 min

Transformation

1. Thaw competent cells on ice.
2. Add 200 µl cells to 10µl liagtion mix
3. Place the mixture put them on ice for 30 min
4. Heatschock 30 sec at 42 °C
5. Place the mixture again on ice for 2 min
6. Add 4 x SOC medium (800 µl)
7. Let them growth for 60 min at 37°C
8. Spin cells down for 5 min at low speed
9. Remove the supernatant, resuspend the cells
10. Spread 100 µl of the cells onto the plates.

PCR

Colonie PCR

Whole plasmid PCR

Mediums

SOB

• 0.5 % (w/v) yeast extract
• 2 % (w/v) tryptone
• 10 mM NaCl
• 2.5 mM KCl
• 20 mM MgSO₄

adjust to pH 7.5 by adding 1M NaOH

LB medium

• 10 g Bacto-tryptone
• 5 g yeast extract
• 10 g NaCl

Total 1 l

M9 minimal medium

10x M9

• 12.8 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 g NaCl
• 1 g NH₄Cl

Total 100 ml

autoclave seperatly

• 10 x M9
• 1 M MgSO₄
• 1 M CaCl₂
• 1 % (w/v) thiamine solution
• 20 % (w/v) glucose

SOC

• SOB
• 20 mM glucose

Used parts from registry

• BBa R0040 P_Tet
• BBa R0051 λ_P
• BBa R0010 P_lac
• BBa R0061 P_lux
• BBa J23100 P_const
• BBa C0061 LuxI_LVA
• BBa C0040 TetR_LVA
• BBa C0051 CI
• BBa C0062 LuxR
• BBa B0015 Double Terminator

Plasmid list

Primer list

Synthesized parts

• LacI_M1
• AlcR (codon optimized)

Characterization of LacI_M1