Golden Gate Assembly protocol - Revision history

Macampbell at 06:07, 3 March 2019

2019-03-03T06:07:53Z

Macampbell at 16:56, 8 June 2016

2016-06-08T16:56:23Z

Macampbell at 16:55, 8 June 2016

2016-06-08T16:55:49Z

Macampbell: /* Protocol Details for GGA */

2016-06-08T16:45:42Z

‎Protocol Details for GGA

Macampbell at 16:44, 8 June 2016

2016-06-08T16:44:09Z

Macampbell at 15:10, 14 September 2012

2012-09-14T15:10:14Z

Macampbell at 14:54, 14 September 2012

2012-09-14T14:54:08Z

Macampbell at 14:30, 14 September 2012

2012-09-14T14:30:05Z

Macampbell: /* Starting wtih Promoter already cloned in a Plasmid */

2012-08-08T13:28:17Z

‎Starting wtih Promoter already cloned in a Plasmid

Macampbell: /* Starting with Boiled then Cooled Oligos */

2012-08-08T13:27:43Z

‎Starting with Boiled then Cooled Oligos

@@ Line 26: / Line 26: @@
 µL dH<sub>2</sub>O <br>
 µL 10X NEB Ligase Buffer<br>
-.5 µL Bsa I high fidelity (HF) restriction enzyme<br>
+.5 µL Bsa I high fidelity (HFv2) restriction enzyme<br>
 <u>0.5 µL T4 DNA ligase from NEB</u><br>
 µL final volume <br> <br>

@@ Line 23: / Line 23: @@
 '''GGA mixture contains:'''<br>
-µL (50 ng) plasmid containing [http://partsregistry.org/Part:BBa_J119137 part J119137]<br>
+µL (50 ng) plasmid containing receiving plasmid such as [http://partsregistry.org/Part:BBa_J119137 part J119137]<br>
 µL dH<sub>2</sub>O <br>
 µL 10X NEB Ligase Buffer<br>

@@ Line 3: / Line 3: @@
 ==Starting with Boiled then Cooled Oligos==
 # You should have already used the [http://gcat.davidson.edu/iGem10/index.html Oligator] to design your oligos for self-assembly into dsDNA. You can take advantage of Oligator's capacity to add the sticky ends onto your oligos, or you can do that yourself. Oligator can also add the BsaI restriction sites onto your DNA which will be cut off by BsaI enzyme in the GGA mixture. <br>
 # Use the protocol for [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html boiling oligos to assemble dsDNA].
-If you don't have time to ethanol precipitate and quantify your DNA, you can use this shortcut:
 * 1 µL cooled oligos.
 * 99 µL water to make a 100X dilution.
@@ Line 12: / Line 13: @@
 <br>
 ==Protocol Details for GGA==
@@ Line 21: / Line 18: @@
 # Get '''two''' new, small microfuge tubes designed to fit into the PCR machine.
 # One tube will be labeled "ligation", the other "plasmid only". Also put your initials on the tubes.
-# 1 µL diluted oligos cooled overnight (''use water instead for "plasmid only" tube''). If you are using [http://partsregistry.org/Part:BBa_J119022 BBa_J119022], use 50 ng in 1 µL volume.
+# 1 µL diluted oligos cooled overnight (''use water instead for "plasmid only" tube'').
-# 9 µL '''GGA mixture''' provided by your instructor.
+# 9 µL '''GGA mixture'''
-# Use the promoter DNA for the ligation tube but use only water for the plasmid only tube. <br> <br>
+# Use the boiled and cooled promoter DNA for the ligation tube but use only water for the plasmid only tube. <br> <br>
 '''GGA mixture contains:'''<br>
-µL (50 ng) plasmid containing [http://partsregistry.org/Part:BBa_J100091 part J100091]<br>
+µL (50 ng) plasmid containing [http://partsregistry.org/Part:BBa_J119137 part J119137]<br>
 µL dH<sub>2</sub>O <br>
 µL 10X NEB Ligase Buffer<br>
@@ Line 33: / Line 30: @@
 µL final volume <br> <br>
-Turn on the PCR machine. Put '''both''' of your tubes into the machine. <br>
+Turn on the thermocycler machine. Put '''both''' of your tubes into the machine. <br>
 Program it for the following cylces: <br>
 * 20 cycles of 37C for 1 minute/16C for 1 minute <br>

@@ Line 28: / Line 28: @@
 µL (50 ng) plasmid containing [http://partsregistry.org/Part:BBa_J100091 part J100091]<br>
 µL dH<sub>2</sub>O <br>
-µL 10X Promega Ligase Buffer<br>
+µL 10X NEB Ligase Buffer<br>
 .5 µL Bsa I high fidelity (HF) restriction enzyme<br>
-<u>0.5 µL T4 DNA Ligase</u><br>
+<u>0.5 µL T4 DNA ligase from NEB</u><br>
 µL final volume <br> <br>

@@ Line 27: / Line 27: @@
 '''GGA mixture contains:'''<br>
 µL (50 ng) plasmid containing [http://partsregistry.org/Part:BBa_J100091 part J100091]<br>
 µL dH<sub>2</sub>O <br>
 µL 10X Promega Ligase Buffer<br>
 .5 µL Bsa I high fidelity (HF) restriction enzyme<br>
 <u>0.5 µL T4 DNA Ligase</u><br>

← Older revision		Revision as of 14:30, 14 September 2012
Line 1:		Line 1:
−	<center>'''Protocol to insert new promoter made into pSB1A2 that contains [http://partsregistry.org/Part:~~BBa_J119044 BBa_J119044~~]'''</center><br> <br>	+	<center>'''Protocol to insert new promoter made into pSB1A2 that contains [http://partsregistry.org/Part:BBa_J100091 BBa_J100091]'''</center><br> <br>

	==Starting with Boiled then Cooled Oligos==		==Starting with Boiled then Cooled Oligos==

@@ Line 14: / Line 14: @@
 ==Starting wtih Promoter already cloned in a Plasmid==
-You can start with [http://partsregistry.org/Part:BBa_J119022 BBa_J119022] which contains a promoter that is flanked by BsaI sites. Use 50 ng of this plasmid for your "insert" DNA to be added to the ligation mixture listed below.
+You can start with [http://partsregistry.org/Part:BBa_J119022 BBa_J119022] which contains a promoter that is flanked by BsaI sites. <br>
 ==Protocol Details for GGA==