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Revision as of 14:51, 20 September 2012 (view source) Jakobmatthes (Talk | contribs) (→PCR) ← Older edit		Revision as of 14:53, 20 September 2012 (view source) Jakobmatthes (Talk | contribs) (→Restriction digest) Newer edit →
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	=== Restriction digest ===		=== Restriction digest ===
		+
		+	'''control digest (10 µl)'''
		+
		+	'''preperative double digest (150 µl)'''
		+
		+	'''plasmid linearization (pRS. 150 µl)'''
	=== PCR ===		=== PCR ===

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Revision as of 14:53, 20 September 2012

Contents 1 Protocols 1.1 Chemo-competent cells 1.2 pGEM Ligation 1.3 Ligation 1.4 Chemotransformation 1.5 Restriction digest 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 (10 µl)

preperative double digest (150 µl)

plasmid linearization (pRS. 150 µl)

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	-	4°C (program finished)

Gel electrophoresis

incl. TAE-Puffer

LB medium

incl. Agarplatten

SOB medium