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From 2012.igem.org

Contents 1 Protocols 1.1 Chemo-competent cells 1.2 pGEM Ligation 1.3 Ligation 1.4 Chemotransformation 1.5 Restriction digest 1.5.1 control digest 1.5.2 preperative double digest 1.5.3 plasmid linearization 1.6 PCR 1.7 Gel electrophoresis 1.8 LB medium 1.9 SOB medium

Protocols

Chemo-competent cells

pGEM Ligation

Ligation for TA-cloning of PCR products

Component	Volume
2X Rapid Ligation Buffer	5µl
pGEM vector	0.5µl (25ng)
PCR product	3.5µl
T4 DNA ligase	1µl (3 Weiss units)

Mix all reagents in a 0.5ml tube. Incubate reaction at 4°C over night.

Ligation

Ligation for digested parts and vectors.

Component	Volume
10X T4 DNA Ligase Buffer	1µl
vector DNA	1µl (20-100ng)
insert DNA	5µl (up to 5:1 molar ratio insert to vector)
T4 DNA ligase	1µl (1 unit)
water	2.5µl

Mix all reagents and incubate at 22°C for 1h.

Chemotransformation

Component	Volume
chemo-competent E. coli	100µl
plasmid DNA	up to 10µl (max. 1/10 of volume)

1. Add plasmid DNA to cell culture.
2. Incubate for 30 min on ice.
3. Heat shock for 90 sec at 42°C.
4. Add 900 µl LB.
5. Let the bacteria grow at 37°C at least for 1 hour.

Restriction digest

control digest

preperative double digest

plasmid linearization

PCR

Component	Volume
Taq/Pfu buffer	5 µl
Taq/Pfu polymerase	1 µl
primer forward	0.5 µl (100 pmol/µl)
primer reverse	0.5 µl (100 pmol/µl)
dNTPs	2.5 µl (200 µM)
template DNA	1 µl
water	36 µl

PCR-Conditions:

Step	Duration	Settings
1	2 min	94°C
2	45 sec	94°C
3	30 sec	gradient or annealing temperature
4	90 sec	72°C
		steps 2-4: 30 cycles
5	7 min	72°C
6	(hold)	4°C

Gel electrophoresis

incl. TAE-Puffer

LB medium

incl. Agarplatten

SOB medium