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Revision as of 14:31, 20 September 2012 (view source) Jakobmatthes (Talk | contribs) (→Protocols) ← Older edit		Revision as of 14:40, 20 September 2012 (view source) Jakobmatthes (Talk | contribs) Newer edit →
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	Mix all reagents and incubate at 22°C for 1h.		Mix all reagents and incubate at 22°C for 1h.
-	=== Transformation ===	+	=== Chemotransformation ===
		+	{| class="wikitable"
		+	|-
		+	! Component !! Volume
		+	|-
		+	| chemo-competent ''E. coli'' || 100µl
		+	|-
		+	| plasmid DNA || up to 10µl (max. 1/10 of volume)
		+	|}
		+	# Add plasmid DNA to cell culture.
		+	# Incubate for 30 min on ice.
		+	# Heat shock for 90 sec at 42°C.
		+	# Add 900 µl LB.
		+	# Let the bacteria grow at 37°C at least for 1 hour.
	=== Restriction digest ===		=== Restriction digest ===
	=== PCR ===		=== PCR ===

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Revision as of 14:40, 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

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

PCR