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Revision as of 15:04, 20 September 2012 (view source) Jakobmatthes (Talk | contribs) (→Ligation) ← Older edit		Revision as of 15:05, 20 September 2012 (view source) Jakobmatthes (Talk | contribs) (→Protocols) Newer edit →
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	! Component !! Volume		! Component !! Volume
	|-		|-
-	| 2X Rapid Ligation Buffer || 5µl	+	| 2X Rapid Ligation Buffer || 5 µl
	|-		|-
-	| pGEM vector || 0.5µl (25ng)	+	| pGEM vector || 0.5 µl (25ng)
	|-		|-
-	| PCR product  || 3.5µl	+	| PCR product  || 3.5 µl
	|-		|-
-	| T4 DNA ligase || 1µl (3 Weiss units)	+	| T4 DNA ligase || 1 µl (3 Weiss units)
	|}		|}
-	Mix all reagents in a 0.5ml tube. Incubate reaction at 4°C over night.	+	Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.
	=== Ligation ===		=== Ligation ===
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	! Component !! Volume		! Component !! Volume
	|-		|-
-	| 10X T4 DNA Ligase Buffer || 1µl	+	| 10X T4 DNA Ligase Buffer || 1 µl
	|-		|-
-	| vector DNA || 1µl (20-100ng)	+	| vector DNA || 1 µl (20-100 ng)
	|-		|-
-	| insert DNA  || 5µl (up to 5:1 molar ratio insert to vector)	+	| insert DNA  || 5 µl (up to 5:1 molar ratio insert to vector)
	|-		|-
-	| T4 DNA ligase || 1µl (1 unit)	+	| T4 DNA ligase || 1 µl (1 unit)
	|-		|-
-	| water || 2.5µl	+	| water || 2.5 µl
	|}		|}
-	Mix all reagents and incubate at 22°C for 1h.	+	Mix all reagents and incubate at 22°C for 1 hour.
	=== Chemotransformation ===		=== Chemotransformation ===
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	! Component !! Volume		! Component !! Volume
	|-		|-
-	| chemo-competent ''E. coli'' || 100µl	+	| chemo-competent ''E. coli'' || 100 µl
	|-		|-
-	| plasmid DNA || up to 10µl (max. 1/10 of volume)	+	| plasmid DNA || up to 10 µl (max. 1/10 of volume)
	|}		|}
	# Add plasmid DNA to cell culture.		# Add plasmid DNA to cell culture.

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Revision as of 15:05, 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 1.5.1 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	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 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