"

From 2012.igem.org

Protocols

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 preparative double digest 1.5.3 plasmid linearization 1.6 PCR 1.7 Gel electrophoresis 1.8 LB medium 1.9 SOB medium 1.10 Genaxxon Plasmid DNA Purification Mini Prep Kit 1.11 Genaxxon Gel Extraction Mini Prep Kit 1.12 Genaxxon PCR DNA Purification Mini Prep Kit 1.13 Qiagen Plasmid Midi Kit

Chemo-competent cells

pGEM Ligation

Ligation for TA-cloning of PCR products

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

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

Ligation

Ligation for digested parts and vectors

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

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

Chemotransformation

Component	Volume
chemo-competent E. coli	100 µl
plasmid DNA	up to 10 µl (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 for at least 1 hour.

Restriction digest

control digest

Component	Volume
Tango buffer 10x	1 µl
XbaI (RE)	0.5 µl
SpeI (RE)	0.5 µl
DNA	1 µl (up to 1 µg)
water	7 µl

preparative 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

TAE buffer 50x

Component	Volume
0.05 M EDTA	18.61 g
1 M acetic acid	60.05 g
2 M Tris	242.28 g
water	1 l

Adjust to pH 8.5.

Gel

Component	Volume
TAE 1x buffer	120 ml
Agarose	1.2 g

Solve agarose in TAE 1x buffer and boil until solution is clear.

Well loading

Component	Volume
PCR product or DNA	5 µl
Loading dye 6x	1 µl

Can be scaled up linearly.

LB medium

incl. Agarplatten

SOB medium

Genaxxon Plasmid DNA Purification Mini Prep Kit

Genaxxon Gel Extraction Mini Prep Kit

Genaxxon PCR DNA Purification Mini Prep Kit

Qiagen Plasmid Midi Kit