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From 2012.igem.org

Revision as of 20:15, 26 September 2012 (view source) Bayke5 (Talk | contribs) (Created page with " {| class="bigtable" |- ! Destination Plasmid DNA !! 500 ng |- | EcoRI-HF || 1 µl |- | EcoRI-HF || 1 µl |- | Pstl || 1 µl |- |10X NEBuffer || 2 5 µl |- |100X BSA || 0.5 µl |...") ← Older edit		Latest revision as of 20:26, 26 September 2012 (view source) Bayke5 (Talk | contribs)
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	{| class="bigtable"		{| class="bigtable"
	|-		|-
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	| EcoRI-HF || 1 µl		| EcoRI-HF || 1 µl
	|-		|-
-	| EcoRI-HF || 1 µl	+	| PstI || 1 µl
	|-		|-
-	| Pstl || 1 µl	+	|10X NEBuffer || 2.5 µl
-	|-	+
-	|10X NEBuffer || 2 5 µl	+
	|-		|-
	|100X BSA || 0.5 µl		|100X BSA || 0.5 µl
	|-		|-
	|H2O || to 50 µl		|H2O || to 50 µl
		+	|-
		+	Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.
		+
		+
		+	Ligate the Upstream and Downstream Parts into the digested Destination Plasmid. {| class="bigtable"
		+	|-
		+	! Upstream Part digestion || 2 µl
		+	|-
		+	| Destination Plasmid digestion || 2 µl
		+	|-
		+	| 10X T4 DNA Ligase Buffer || 2 µl
		+	|-
		+	| T4 DNA Ligase || 2 µl
		+	|-
		+	|H2O || 11 µl
	|-		|-

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Latest revision as of 20:26, 26 September 2012

Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes. Ligate the Upstream and Downstream Parts into the digested Destination Plasmid. {| class="bigtable"

Destination Plasmid DNA	500 ng
EcoRI-HF	1 µl
PstI	1 µl
10X NEBuffer	2.5 µl
100X BSA	0.5 µl
H2O	to 50 µl
Upstream Part digestion	2 µl
Destination Plasmid digestion	2 µl
10X T4 DNA Ligase Buffer	2 µl
T4 DNA Ligase	2 µl
H2O	11 µl